Digital PCR To Quantify Evolution Of Mycobacterium Tuberculosis SNP Proportion During In Vitro Competitive Assay
1Centre International de Recherche en Infectiologie, INSERM U1111, Université de Lyon, Lyon, France; 2Plateforme DTAMB, CNRS, Université Lyon 1, Villeurbanne, France; 3Hospices Civils de Lyon, Institut des Agents Infectieux, Laboratoire de bactériologie, Lyon, France
Background: Tuberculosis caused by Mycobacterium tuberculosis (MTB) complex remains a deadly infectious diseases worldwide. MTB infection results in a variety of disease manifestations and epidemiological success: latent asymptomatic infection and/or progression to pulmonary or extra-pulmonary injuries, with an array of severity symptoms. To assess the performances of genetic variants, we used in vitro competitive assays with and without selection pressure and evolution of variant proportion was evaluated by digital PCR.
Method: Isogenic clones, differing by only one SNP in locus involved in the biosynthesis of lipids from the mycobacterial outer membrane, were mixed at various ratio to obtain increasing proportion of the mutation in the bacterial mixture. MTB mixture grew in liquid medium with or without rifampicin, a first line antibiotic, during 4 weeks; MTB DNA was extracted and proportion of mutation at the end of the experiment was evaluated by digital PCR.
Results: Without antibiotic pressure we did not obtained significant differences in growth curves and digital PCR did not reveal significant evolution of mutation proportion in the bacterial mixture. Conversely, under rifampicin pressure we observed a better growth of MTB with the increase of the mutation in the bacterial mixture and digital PCR revealed a significant selection of this SNP during rifampicin exposure.
Conclusion: This study highlights a selective advantage of the SNP studied during rifampicin exposure and validates digital PCR as a tool to explore MTB community dynamics in in vitro models.
Development of a Strain-specific Droplet Digital PCR (ddPCR) for Detection and Quantification of a Probiotic Strain Bifidobacterium animalis (BAN) in Feed.
BIOMIN Research Center, Technopark 1A, 3430 Tulln, Austria
The World Health Organization (WHO) defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Nowadays, the use of probiotics in animal nutrition to provide health benefits is common and widely accepted.
The use of real-time PCR is very common in the food industry, given that a method was determined to remove target DNA from non-viable cells through optimized DNA extraction. Nevertheless, the presence of inhibitors in feed samples can compromise the results and efficiency of this molecular technique. It has been demonstrated that the Droplet Digital PCR (ddPCR) in comparison to real-time PCR has higher tolerance to PCR inhibitors. In this study, a strain-specific ddPCR assay was developed for identification and quantification of a probiotic strain Bifidobacterium animalis (BAN) when added to poultry feed.
Therefore, the following chemical and physical parameters were taken into account: the optimal annealing temperature, concentration of primers and probe, and number of PCR-cycles. Ranges from 56.6°C to 62.6°C, 100 nM to 1000 nM, and 40 and 50 cycles were investigated, respectively. Optimal parameters resulted to be 59°C, 500 nM primers and 400 nM probe concentration, and 50 cycles. The linearity of the optimized ddPCR assay was established from the undiluted DNA until 1:10000dilution. For result validation, actual ddPCR results were compared to calculated results based on initial DNA concentration. The relation between expected results and ddPCR results was linear (R2=1), the efficiency (E) of the method was 99,98%. To warrant unbiased and repeatable ddPCR assay runs, purified BAN DNA was used.
To finalize the development, the Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantification (LoQ) for the assay were determined using poultry feed free of BAN, and spiked with different concentrations from 2,0E+06 CFU BAN/g feed to 1,91E+0 CFU BAN/g. Results showed a LoB of 3,07E+02 copies BAN/g feed, a LoD of 5,42E+02 copies BAN/g feed and a LoQ of 1,57E+03 copies BAN/g feed.
This ddPCR method is a valid method for quality control of animal feed supplemented with probiotic bacteria, to ensure appropriate inclusion rate of the product.
Design of a Digital PCR Assay for the Simultaneous Quantification of 14 Genetically Modified Soybean Lines in a Single Reaction
BIOTECON Diagnostics GmbH, Germany
The cultivation of genetically modified (GM) crops has increased steadily over the last years and their presence in food products is regulated in many countries. The European Union (EU) has set a labelling threshold for food products containing more than 0.9% of authorized genetically modified organisms (GMO) in Regulation 1829/2003. This threshold is based on the total concentration of GM-events per ingredient.
The most common method for GMO quantification is standard-curve based real-time PCR. However, this method is usually limited to a single GMO event per assay. Since the number of authorized GMOs in the food market is increasing, faster and cost-efficient detection methods need to be developed to make monitoring feasible. Digital PCR is a suitable alternative to real-time PCR which allows multiplexed GMO quantification without the need for a standard curve. An additional advantage is the higher tolerance towards inhibitors present in plant material.
A digital PCR assay for quantification of all 14 soybean events currently authorized in the EU in a single reaction was developed by BIOTECON Diagnostics. In this multiplex assay all 14 GM soybean lines are quantified simultaneously in the FAM channel by amplification of their event specific sequences. The reference gene lectin is quantified in the HEX channel and the total GM soybean content of a sample is expressed as ratio between the species-specific reference gene and the sequences of the GM-events.
The assay is able to precisely and reliably quantify GMO contents at the regulatory thresholds of 0.9% and 0.1% which are stated in EU Regulation 1829/2003 and the German “GMO-free” label specifications laid down by Lebensmittel ohne Gentechnik e.V. (VLOG), respectively. Also the overall GMO content can be quantified in a single reaction from mixtures of events since all GM events are detected in one channel. The assay did not amplify DNA from plant species other than soy and is applicable to various relevant sample matrices. This was shown by spiking food products with DNA extracted from GM positive certified reference materials. The assay robustly separates positive and negative droplets in the digital PCR with only a low amount of rain for all events.
We successfully achieved quantification of all 14 soybean events in a single reaction. Using this new digital PCR assay quantification of approved soy GMOs is faster and cheaper than individual quantification by real-time PCR. Our assay fulfills all criteria to be used for routine monitoring of GM soybeans in food products.
A Rapid, Simple, High-throughput Compatible Approach to Generating CRISPR/Cas9 Knock-out Cell Lines
Reagent R&D, Life Sciences Group, Bio-Rad Laboratories,
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock-out cell lines are cumbersome, labor intensive and comprise of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of Droplet Digital™ PCR (ddPCR™), high-resolution melt analysis (HRM), and western blot analysis to generate 3 knock-out cell lines in under 5 weeks.
The Good, the Bad and the Ugly: Selective single cell isolation
1MMI - Microscopic Single Cell Isolation, Germany; 2University of Applied Sciences, Weihenstephan-Triesdorf, Germany
The isolation of single cells is an essential pre-requisite for many research projects in pathology, oncology or forensics.
The MMI CellEctor is designed to facilitate selective isolation of single cells in suspensions.
This study demonstrates the development of the novel Shake Mode feature which now enables for the efficient but gentle uptake of living adherent cells. Thus, cells can be used for all kinds of downstream analysis as well as for further cultivation.
As an application example, we show single-cell mRNA profiling using CTCs from liquid biopsies.
A SMARTer Solution To Stranded Single-Cell RNA-Seq
1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA
Our SMARTer® NGS reagent portfolio has long included highperformance, cutting-edge solutions for RNA sequencing (RNA-seq). With the growing need for low-input and single-cell NGS library prep solutions, we see that researchers recognize the value in revealing transcriptome profiles from damaged cells as well as noncoding information from extremely low cell numbers (1–1,000). While we have previously released several industry-leading products that push the limits of sensitivity and reproducibility in RNA-seq from ultra-low inputs as well as single cells (SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing and SMART-Seq HT Kit), they both generate transcriptome profiles from mRNA only. Oligo(dT) priming is a very efficient way to capture the transcriptome, with minimal uninformative reads (e.g., from rRNA contamination), but it does not provide a complete view of the transcriptome, as only the polyadenylated fraction can be captured. In addition, for oligo(dT)-primed cDNA synthesis to generate high-quality libraries, one needs to start with high-quality, intact RNA, which excludes the use of this technology with samples damaged or degraded due to the nature of the processing (e.g., FFPE samples) or method of isolation. Additionally, these earlier single-cell kits do not preserve strand-oforigin information. All of these factors motivated the development of the SMART-Seq Stranded Kit, which allows for generation of sequencing-ready, stranded Illumina® libraries directly from 1–1,000 sorted cells or an equivalent amount (10 pg–10 ng) of purified total RNA of any quality. This kit integrates an innovative technology, already incorporated in our SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, which enables removal of ribosomal cDNA following cDNA synthesis, as opposed to direct removal of corresponding rRNA molecules prior to reverse transcription. Since cDNA synthesis in the SMART-Seq Stranded Kit relies on random priming, rRNA is also captured, and remov al of the resulting cDNA prior to sequencing is essential. The SMART-Seq Stranded Kit protocol can be completed within seven hours, and a convenient pooling option for inputs between one to ten cells facilitates greater ease-of-use by minimizing the number of samples being handled (Figure 1).
TATAA Alu Repeat qPCR Assays – a Tool for Contamination Control and Quality Assessment in Single Cell and Cell-Free DNA Analysis
TATAA Biocenter, Sweden
Laboratory reagents, including PCR master mixes, primers and probes, are commonly contaminated with residual human genomic DNA (gDNA) from the manufacturing process. For conventional measurements, the contamination can typically be considered negligible, but poses a significant, if not detrimental, problem in nucleic acid analysis applications where low-abundant targets will be studied, such as single cell and rare mutation analysis.
To test the purity of reagents TATAA Biocenter has developed ultra-sensitive qPCR assays targeting human specific Alu repeats. Alu repeats are short sequences of about 300 bp dispersed across our genetic code, summing up to more than one million copies per genome. This corresponds to more than 10% of our hereditary material, rendering Alu repeats the most abundant sequence of the human genome. Therefore, the TATAA assays will reliably detect even minuscules amounts of human gDNA contamination.
The TATAA Alu assays are designed to target multiple copies to generate reliable results also for very low amount of human gDNA. Alu assays are available that produce different amplicon lengths (60, 135 and 187 bp), which makes it possible to assess also the length distribution and integrity of the gDNA present. For example, via analysis of a sample with TATAA ALU-60 and TATAA ALU-187 assays, the level and fraction of cell-free DNA (cfDNA) in liquid biopsy samples can be assessed.
The ALU quality control panel was in part developed for the CANCER-ID consortium, developing standard operating procedures for liquid biopsy, and as quality control tool for ring trials run in collaboration with the SPIDIA consortium standardizing the preanalytical workflow.
SureCellⓇ ATAC-Seq Library Prep Kit: A New Solution for Single-Cell ATAC-Seq using Bio-Rad’s Droplet Digital Technology
Bio-Rad Laboratories, Digital Biology Center, Pleasanton CA
Cellular states and the activation of gene expression are governed by functional interactions between transcription factors and DNA regulatory elements. scATAC-seq (single cell Assay for Transposase Accessible Chromatin usingsequencing) is a powerful tool to interrogate chromatin accessibility genome wide at single-cell resolution. To enable the widespread and facile application of this approach, we developed a scATAC-seq platform using Bio-Rad’s Droplet Digital technology. Here, we describe a cell capture strategy that enables processing of thousands of cells per experiment with cell utilization efficiencies greater than 50%. We also show improved sensitivity and signal to noise relative to a previously published gold standard. The method is a push button workflow that can be comfortably completed in less than a day with only five user steps. We apply this technology to profile the epigenomes of tens of thousands of single cells across normal hematopoiesis and in response to immunological stimuli to discover a large repertoire of lineage- and stimulus-specific non-coding regulatory elements and transcription factors. Altogether, we show that these rich data sets and experimental tools provide a new platform for discovering regulatory differences across cells within heterogeneous tissues enabling the discovery of cis- andtrans-acting effectors of cellular differentiation and disease.
Single-cell studies: Focus on reverse transcription
1Laboratory of Gene Expression, Institute of Biotechnology CAS, Czech Republic; 2TATAA Biocenter, Sweden
In the field of gene expression research, the reverse transcription (RT) is often the initial enzymatic reaction when processing the samples. However, it is known that the reaction output is far from ideal, as it relies on the choice of priming strategy, template and reverse transcriptase used.
The aim of this study was to compare the sensitivity, accuracy, reproducibility and yield of 11 commercially available reverse transcriptases with varying origin, RNase H activity and price. In consequence, two selected RTases were tested for further practical parameters, conducted on high-throughput platform. All measurements were executed under two priming strategies – routine RT-qPCR mixture of primers or reduced oligo(dT) concentration common in single-cell RNA-Seq protocols. Both single-cell and 100-cell bulk reactions were investigated.
Despite each reverse transcriptase performed with great accuracy and reproducibility, the ability to capture rare transcripts varied considerably. Similarly, differences were observed in reaction yields, where SuperScript IV and Maxima H- enzymes performed with significant efficiency. Consequent measurements confirmed Maxima‘s level of performance in single-cell RT-qPCR experiment, such as higher reaction positivity rate, signal strength or cluster separation. Methodical options of RT priming were also remarked, reporting considerable differences between priming alternatives. In final, the position of PCR targeted sequence has a minor role in its detection efficiency. Still, targeting of RNA 3’end regions was found to be the most reliable option.
Our results provide thorough comparison of available reverse transcriptases for both routine RT-qPCR and RNA-Seq applications, but also discuss the impact of RT priming strategy or template abundancy on reaction outcome. The choice of better RTase makes a substantial change in the outcome of RNA experiment. Practicality of PCR primer design is associated. Thus, this work serves as a guideline for improvement in RNA focused research.
High-througput, full-length, single-cell RNA sequencing
1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA
Since the emergence of next-generation sequencing (NGS), the importance and demand for single-cell analyses have risen rapidly. Single-cell RNA-seq can be used to generate data for analysis of differential gene expression, alternative splicing, gene fusions, and so on, allowing for cell profiling in detail. As a result, this technology has been gaining prominence not only in basic research fields, but also in the clinical space. Therefore, automation systems that can process many single cells regardless of cell type or size, and that provide high-quality data with high sensitivity, reproducibility, and fulllength sequence information are desired. Our method presented here for full-length RNA-sequencing the SMARTer™ ICELL8® automation system meets the demands described above. Our SMARTer ICELL8 system is an automated platform specialized for single-cell analysis. Unlike other automation systems, the SMARTer ICELL8 system utilizes a simple dilution method with a large aperture dispenser for single-cell isolation, enabling users to characterize many types of cells in a small amount of time. In addition, the SMARTer ICELL8 system provides the capacity to image and select cells for downstream analysis on a well-by-well basis, such that >1700 single cells can be analyzed at once. Our method for full-length RNA-sequencing the SMARTer ICELL8 system will enable users to perform all reactions required for Illumina® NGS library preparation in the ICELL8 chip, including cDNA synthesis and PCR amplification, so that the output from the chip is a sequencing-ready library. The most important feature of our approach is that the output library contains full-length sequence information as opposed to only capturing RNA 3’ ends (data typically used for differential gene expression analysis). Along with incorporating SMART® template-switching technology to ensure production of high-quality cDNA, our method for full-length single-cell RNA-seq with the SMARTer ICELL8 system enables various analyses to be performed with high sensitivity and reproducibility.
Clinicаl Аpplicаtion of Next Generаtion Sequencing Technology Methods
USEK, Lebanon (Lebanese Republic)
The humаn body cаn be regаrded аs а constаnt temperаture fermenter for intestinаl microbes. The rich nutrients in the intestines аre the culture medium for bаcteriа. In fаct, we only hаve 1% The genes аre from pаrents, аnd the remаining 99% аre from microbes in the externаl environment (Bolcа et аl., 2013). They аre the bаcteriа in our body аnd in the intestines. Аt present, there аre more thаn 2,000 kinds of intestinаl microorgаnisms identified, аccounting for 1-3% of our body weight. In terms of quаntity, intestinаl microbes аlso hаve аn аbsolute аdvаntаge. When they аre mаny, they cаn reаch 10 times of humаn cells, but in generаl, they аre аt leаst equivаlent to the number of humаn cells. The lаtest estimаte is аbout 1.36 times thаt of humаn cells (Bolcа et аl., 2013).
The Humаn heаlth is insepаrаble from microbes. These lаrge numbers of microbes thаt breed on our body or in the body hаve аlreаdy tаken our body аs their "coloniаl": when the microbes settled in our bodies, they begаn to do whаt а colonizer should do: build а house, Eаting, breeding, resisting foreign enemies or invаding other florа (Аbubucker et аl., 2012).
In fаct, while аdаpting, microbes аre constаntly chаnging their living environment. Their excretа or metаbolites not only аffect their own survivаl, but аlso chаnge the metаbolism of humаn cells аnd the expression of genes. Once the gut microbiotа is out of tune, we will аlso be involved in them аnd get involved in their disputes; then inflаmmаtion, obesity аnd even cаncer come one аfter аnother (Greenblum et аl., 2012).
Bаriаtric surgery is а surgicаl treаtment for obesity. This type of surgery hаs proven to be more effective in treаting obesity thаn lifestyle chаnges, dieting аnd drug weight loss. Weight loss surgery аchieves the effect of inducing weight loss by chаnging different pаrts of the gаstrointestinаl trаct.
NGS is аlso known аs high-throughput sequencing, including second-generаtion mаssively pаrаllel sequencing аnd three-generаtion single-molecule sequencing (Mаrdis, 2008). There аre still mаny prаcticаl problems in the routine аpplicаtion of NGS technology in clinicаl tests. The complexity of clinicаl specimens mаy result in too little pаthogen informаtion аnd dаtа loss. Pаthogen dаtа mаy be mixed in the normаl gut microbiotа аnd it is difficult to distinguish (Keller et аl., 2014).
There аre no different specimens yet. Uniform provisions for pre-sequencing processing аnd pаrаmeter setting; high-throughput sequencing will generаte lаrge аmounts of dаtа, requiring huge dаtа аnаlysis. If there is а lаck of locаl dаtаbаses, the cost аnd time of computing work is difficult to control; for gut microbiotа, sequencing of genomic dаtа; the relаtionship with it is still uncleаr. Аccess to genomic informаtion аlone is not sufficient to clаrify gut microbiotа аnd the process of its development. Аccurаte аnаlysis аnd dаtа mining аre needed to аchieve precise treаtment аnd intervention.
Full-length 16S rRNA Sequencing Combined with the Illumina Barcode Structure Allows a Deeper Insight into Strains Present in Stool Samples
Core Facility Microbiome/NGS, ZIEL - Institute for Food and Health,Technische Universität München, Germany
Background: Based on their ubiquitous presence, evolutionary stability and changes in its variable sequences, genes of the small ribosomal subunit are the most commonly used phylogenetic marker. Nevertheless, 16S rRNA analyses are mostly performed by short amplicon sequencing for the ribosomal gene of the small subunit (SSU) and are thereby limited. An easy, cheap and user-friendly method for full-length SSU rRNA sequencing should, therefore, be developed.
Results: We described a simplified method for full-length SSU rRNA sequencing using the Illumina MiSeq platform. The method has been improved such that the standard Illumina 16S barcode primers can be used and only adaptor primers in addition, which are low in cost are needed. Moreover, the protocol is shortened and tested for a better reproducibility compared to past proposals.
Conclusion: By using this approach, deeper insights into species and strains present in an unknown sample are possible. The method allows an easier data evaluation and is cost efficient because it can be easily adapted by all laboratories that already sequence amplicons with Illumina’s standard protocol by just adding a limited amount of novel primer.
Simple Whole Genome Amplification Technique Developed on the Basis of SD DNA Polymerase
Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number of genomic DNA; WGA provides the only possibility to work with single-cells genomes so far. There are a number of WGA methods that have been developed. Degenerate Oligonucleotide-Primed PCR (DOP-PCR) is one of the simplest, fast and inexpensive WGA techniques. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an Improved DOP-PCR with enhanced WGA performance (iDOP-PCR) based on a Bioron’s novel DNA polymerase with a strong strand-displacement activity (SD polymerase, Patent US 9,896,671).
Several commercially available WGA kits were compared with the newly developed Bioron’s iDOP-PCR One Step WGA Kit. The amplified wgaDNA libraries were evaluated by different ways including NGS data.
iDOP-PCR WGA provided comparable or better quality of the amplified DNA libraries compared to the other WGA methods tested, the workflow is more simple, user-friendly and not labor-intensive. Thus, the new technique based on SD-polymerase can be used as a simple and convenient alternative for the Single Cells WGA in research and clinical practice.
Tackling Contaminants In Skin Microbiome Research
1Institute of Environmental Medicine, UNIKA-T, Augsburg, Germany; 2ZIEL - Institute for Food & Health, Freising, Germany
The skin microbiome is of interest as several skin diseases as atopic eczema are associated with a dysbalance in the microbiome. However, skin microbiome analysis is error-prone due to a low amount of microbial DNA in skin samples which increase the risk of contamination.
This has been confirmed by next-generation sequencing of the 16S rRNA of a dilution series of a synthetic bacterial mock community consisting of seventeen different species. The lower the starting material, the more OTUs appeared in the analysis which were not included in the mock community. A major contamination source for the samples are DNA extraction kits. Therefore, we used different DNA extraction kits to obtain DNA of skin swabs taken from adjacent skin site. The 16S NGS results show that samples cluster by DNA extraction kit instead of study participant, emphasizing the impact of contaminants from DNA extraction kits on the result in skin microbiome research.
In order to analyze skin microbiome data in a more accurate way, especially to optimize with objective criteria the handling of contaminations,we have developed a computational platform, MicrobIEM. This tool for microbiome analysis has a graphical interface. MicrobIEM facilitates the identification and exclusion of contaminating OTUs and samples in a user-friendly way based both on objective criteria and prior information. Remaining samples can be selected dynamically from the given meta-information for further analysis including the calculation of alpha and beta-diversity measurements and the taxonomy. Advanced statistical methods are used to allow testing the robustness of correlations with the meta-information even with a low number of samples.
In summary, our results and bioinformatics computational platform would allow better handling of contaminations in skin microbiome analysis.
Microsatellite Instability Analysis and NGS with Fragmented Sample Types
Promega Corporation, United States of America
Introduction: A significant hurdle to using fragmented DNA for genomic studies is obtaining a sample of sufficient quantity and quality for rigorous downstream applications like NGS. Having effective tools to isolate, characterize, and analyze fragmented DNA containing samples, such as circulating cell free DNA (ccfDNA) and FFPE tissues, can prevent downstream failures, ultimately saving hours of work and precious samples. Here we present optimized methods for use with even highly fragmented DNA samples. Using this toolset, we demonstrate successful NGS and microsatellite instability (MSI) workflows using matched FFPE tissues and plasma samples. Methods: Plasma and FFPE tissue samples were obtained from three individuals with colorectal adenocarcinoma. DNA was isolated with Promega’s Maxwell® RSC Instrument using the Maxwell® RSC FFPE DNA Kit for FFPE tissues and the Maxwell® RSC Circulating DNA Kit with the large volume custom protocol for plasma. DNA was then quantified with the ProNex® DNA QC Assay. Following quantitation, MSI analysis and NGS library preparation using the TruSeq Custom Amplicon Low Input Kit from Illumina was performed. NGS libraries were checked for size and quantity and then sequenced on the MiSeq® System (Illumina). Results: Full MSI profiles were obtained from DNA obtained from both ccfDNA and FFPE samples from each individual. Following successful determination of MSI-status, NGS libraries were produced from each sample. Sequencing of these libraries produced mean amplicon read depth greater than 3000x and mean coverage uniformity greater than 90%. In addition to excellent sequencing quality metrics, variants in mismatch repair genes identified in FFPE samples were also detected in matched plasma samples. Conclusions: Proper molecular tools and assays are essential to success in exacting downstream applications like NGS and multiplex PCR. This work introduces streamlined methods for DNA isolation, library preparation, and multiplex microsatellite instability analysis from fragmented sample types and demonstrates their effective use with matched FFPE and ccfDNA samples.
RiboPOOL: An Affordable Custom/Ribosomal RNA Depletion Solution Against Any Species for RNA Sequencing
1siTOOLs Biotech GmbH; 2IMGM Laboratories GmbH; 3University of Würzburg; 4University of Bayreuth
With the rising use of RNA Sequencing (RNA-Seq) with Next-Generation Sequencing (NGS), there is a growing demand for reagents that increase output and reliability of sequencing data. Ribosomal RNAs (rRNAs) occupy more than half of total RNA sequencing reads. For sensitive detection of scientifically relevant RNAs, rRNAs are removed either by physical depletion methods using rRNA-specific probes or by isolating relevant coding RNAs through poly-A-tail affinity enrichment. Current commercial rRNA removal kits are costly and limited to well-studied species. riboPOOLs developed by siTOOLs Biotech in partnership with IMGM Laboratories and scientists from University of Würzburg and Bayreuth, present an affordable and flexible solution that gives scientists the freedom to deplete rRNAs or other custom abundant RNAs from any species. Composed of high complexity pools of optimally-designed biotinylated DNA probes, riboPOOLs specifically associate with cytoplasmic and mitochondrial rRNAs, enabling their removal with magnetic streptavidin-coated beads. Human and mouse riboPOOLs were demonstrated to outperform current commercial rRNA depletion kits (Ribo-Zero, Illumina) in RNA-Seq experiments, depleting ~10% more rRNA while leaving other RNA intact. riboPOOLs have been made against a diverse array of organisms including Planarian, Silkworm, Drosophila, Arabidopsis and Bacteria. Furthermore, riboPOOLs can be tailored to deplete abundant tissue-specific mRNAs, such as globin in red blood cells, making it applicable for NGS-based clinical diagnostics.
Sample Quality Control of Long Read Sequencing and Low Input Libraries
1Agilent Technologies, USA; 2Agilent Technologies, Germany
Long-read sequencing and miniaturization of library preparations are becoming increasingly common as new next-generation sequencing workflows are developed. Traditional quality control methods do not provide the required sizing accuracy of DNA greater than 50kb or the sensitivity allowing for sample conservation during the quality control steps. The Femto Pulse System by Agilent works to streamline quality control by separating gDNA up to 165kb in as little as 70 minutes, down from the 16+ hours required for traditional agarose PFGE. While the unparalleled sensitivity of the Femto Pulse allows for preparation of low input NGS libraries prepared from cfDNA, RNA, and miniaturized traditional DNA NGS libraries. Quality control metrics such as the RNA Quality Number (RQN) and user defined Genomic Quality Number (GQN) aids in the determination of sample quality. This poster shows use of the Femto Pulse system in large gDNA separation and low input library preparation with subsequent analysis features highlighted.
A Guide for Sample Processing to Determine Novel Mitochondrial Genomes by Next-Generation Sequencing
1University of Veterinary Medicine, Vienna, Austria; 2Core Facility Molecular Biology, Center for Medical Research, Medical University Graz, Graz, Austria; 3Department of Behavioural Biology, University of Vienna, Vienna, Austria
Recent studies suggest that the functional repertoire of the mitochondrion had been underestimated. The organelle contains its own genome, the circular mitochondrial DNA (mtDNA), and communicates by anterograde and retrograde signaling with the nucleus, thus mastering metabolism, energy production, cellular signaling and innate immunity. Particularly, the mitochondrial single-stranded 7S DNA  and the mitochondrial derived peptides  representing key features of mitochondrial physiology emerged as topics of mitochondrial research. Here, we describe a simple, efficient and highly reproducible lab protocol for next-generation sequencing (NGS) of novel mammalian mtDNAs.
Without mtDNA enrichment, NGS analysis of the total cellular DNA results in 0.9% mtDNA reads (preliminary data). One option to lower financial expenditure caused by sequencing unwanted sequences of nuclear origin is to deplete linear nuclear enzymatically. It is reported that digestion with the Plasmid-Safe ATP-dependent DNAse followed by multiple displacement amplification results in a mtDNA proportion of up to 30% . Generally, this approach is hampered by the recent finding of extrachromosomal circular DNA (eccDNA) constituting a considerable amount of the circular fraction of cellular DNA . It is currently unclear whether the limitation also extends to platelet DNA, a template type naturally depleted of nuclear DNA. Alternatively, immunoisolation by magnetic beads coupled to an antibody against the mitochondrial outer membrane-protein TOM22 offers one of the most promising ways to increase the proportion of mitochondrial reads (up to 99%,). A similar level of sequence purity can be achieved by long-range PCR amplification primed by back-to-back oligonucleotides, the state-of-the-art approach to identify (human) mitochondrial genomes by NGS technology .
Here we address the issue of designing universal mitochondrial back-to-back primers for de novo sequencing of mtDNA across mammalian phylogeny tocombine cost-efficiency of NGS analysis with a reduced time expenditure for primer design.
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Real-time Genotyping PCR From Blood And Blood Plasma Samples
1Chair of Organic Chemistry / Cellular Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany; 2myPOLS Biotec GmbH, Technologiezentrum Konstanz, Blarerstraße 56, 78462 Konstanz, Germany, www.mypols.de
Blood and blood plasma specimen are known to contain some of the most prominent inhibitors for DNA polymerases. Thereupon most diagnostic applications are based on isolated and purified nucleic acids. This strongly requires isolation and purification of DNA from the blood samples and removal of inhibitory compounds such as hemoglobin and immunoglobulin. Only afterwards, the samples can be analyzed in the respective PCR test. The necessity of the isolation and purification step renders the analysis more cost, labour and time intensive, is error prone and hampers throughput. Noteworthy, this isolation and purification step is currently higher charged in the German health-care system than the PCR of the genotyping test itself.
Based on the vast experience of myPOLS Biotec in the field of DNA polymerase engineering we were able to develop a novel system that enables real-time qPCR reliable analysis directly from blood samples without the requirement of isolation and purification of the DNA. On the poster, we present data of reliable real-time qPCR-based genotyping analysis of direct from certified blood samples on Factor II and Factor V blood samples employing different TaqMan and SimpleProbe assays. myPOLS Biotec now provides kits including this PCR innovation as research-use-only kits (www.mypols.de/directblood-genotyping-pcr-kit). Currently we are further advancing our developments and are planning to provide this innovation as SNP-specific in vitro diagnostic-Kits within the on-going year i.e., especially for Factor II, Factor V, lactose intolerance, HFE, and HLA-B27.
Additionally, we are currently developing advanced real-time PCRs directly from plasma samples for liquid biopsy in collaboration with the University of Konstanz and show first results for the detection of the cancer mutation BRAF V600E.
Considerations and Quality Controls when Analyzing Cell-free DNA
1Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90 Gothenburg, Sweden; 2Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.; 3Respiratory Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.; 4Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden; 5Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA; 6Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden.
Analysis of circulating cell-free DNA (cfDNA) is a promising biomarker in cancer, prenatal testing, transplantation medicine and beyond. Ultrasensitive technologies enable detection of low (< 0.1%) allele frequencies, a pre-requisite to fully utilize the potential of cfDNA for these applications. Reliable cfDNA analysis requires that the entire liquid biopsy workflow is carefully optimized. Here, we illustrate important considerations for cfDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, both DNA fragmentation and the target sequence is shown to affect cfDNA assay performance. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.
Analysis Of Cell-free Tumor DNA Using SiMSen-Seq
1Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden.; 2Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.; 3Sahlgrenska Cancer Center, Department of Laboratory Medicine, Sahlgrenska Academy at University of Gothenburg.; 4Department of Laboratory Medicine, Biobank Core Facility, University of Gothenburg.; 5Biobank West, Sahlgrenska University Hospital, Sweden.; 6Respiratory Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
Analysis of circulating cell-free tumor DNA (ctDNA) in liquid biopsies offers new means for early cancer detection, real-time monitoring of treatment efficiency, and the discovery of relapse. Despite its potential use, ctDNA remains challenging to detect and to quantify as it represents only a small fraction of total plasma circulating cell-free DNA. We have developed an ultrasensitive sequencing technology, SiMSen-Seq, that allows allele frequencies < 0.1% to be detected, using several kilobases of DNA. SiMSen-Seq is simple to perform, flexible in multiplexing and requires minimal DNA input. SiMSen-Seq allows detection of variant alleles with easy customization of library content and a protocol that can be implemented in any molecular biology laboratory. Here, we present how SiMSen-Seq can be implemented in a liquid biopsy workflow, including sample collection, cell-free DNA extraction, sequencing and finally bioinformatics to quantify extremely lowly prevalent disease-specific mutations.
Performance Evaluation of Novel Protocols for Small RNA Sequencing In Biofluids
1Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic; 2Faculty of Chemical Technology, University of Chemistry and Technology, Prague, Czech Republic; 3Faculty of Science, Palacký University, Olomouc, Czech Republic; 4TATAA Biocenter, Gothenburg, Sweden
Circulating cell-free microRNAs are promising candidates for minimally invasive clinical
biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Small
RNA-sequencing (sRNA-Seq) has become the method of choice for the discovery of novel
miRNA biomarkers due to its high sensitivity and specificity, wide dynamic range, capacity to
discover new miRNAs and quantify expression of miRNA isoforms. However, numerous
technical biases associated with the small RNA-Seq library preparation hamper accurate
and reliable quantification. Recently, several new commercial kits for small RNA library
preparation claiming enhanced performance have been launched. However, an independent
evaluation of their performance was missing. Here, we present a benchmarking study
comparing six commercial kits, covering all of the currently available technical approaches
for the small-RNA library preparation. These include standard ligation of two defined
(Lexogen, Norgen), randomized (BiooScientific) or UMI-containing (Qiagen) adapters as well
as novel bias reducing approaches based on poly-A tailing (Takara) and single-adapter
ligation and circularization (Somagenics). We have used standardized human plasma
sample and set of synthetic spike-in miRNAs to evaluate several performance parameters
with focus on the utility of the kits for microRNA profiling from biofluids (sensitivity, tendency
to generate artefacts, spectrum of detected isomiRs, accuracy, false-positivity, ligation and
PCR bias) as well as practical parameters relevant for general users (level of multiplexing,
laboriousness of the protocol, time-consumption). Thus, this work can serve as a
comprehensive guideline for the choice of small-RNA library prep kit based on users´
Taqman Advanced Mirna Cdna Synthesis Kit To Simultaneously Study Expression Of Mirna And Mrna From Serum Samples
Thermo Fisher Scientific, United Kingdom
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. The interplay between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA can be valuable in understanding molecular mechanisms that may potentially have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse transcribe and pre-amplify miRNA and mRNA from sample limiting serum research samples using TaqMan® Advanced miRNA cDNA synthesis Kit.
Taqman Advanced Mirna Cdna Synthesis Kit To Simultaneously Study Expression Of Mirna And Mrna From Serum Samples
New Developments in Nucleic Acid Sample Quality Control
1Agilent Technologies, Germany; 2Alacris Theranostics, Germany
Quality control (QC) of RNA and DNA samples is key for the success of any downstream experiment. Especially, Next Generation Sequencing (NGS) developed to a powerful tool in almost all genetic research and diagnostic areas. Since the downstream applications are often time-consuming and expensive, tight QC steps are required to avoid a “garbage in-garbage out” situation. The ideal QC solution is easy-to-use, economical and provides fast and unambiguous results also for very low concentrated samples. Nucleic acid quality assessment can be standardized using automated electrophoresis systems to ensure that samples are “fit for purpose”. This poster exhibits the latest developments in nucleic acid sample QC and gives application examples – from RNA to Cell-free DNA (cfDNA) - evaluated with an Agilent 4150 TapeStation system. Cell-free DNA (cfDNA), gain more and more importance in the context of cancer research. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. Dependent on preanalytical sample treatment or extraction method, cfDNA samples may contain high molecular weight DNA fragments e.g. genomic DNA contaminations. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. For the objective quality evaluation of gDNA and RNA, the quality scores DNA integrity number (DIN) for gDNA and the RNA integrity number equivalent (RINe)for RNA can be assessed providing numerical values from 1 (degraded) to 10 (intact) for classification of samples.
Plasma cfRNA Profiling using the NanoString nCounter® Low RNA Input Assay
NanoString, United States of America
Liquid biopsy, as a blood-based diagnosis with the advantages of easy access, less invasion, and repeatability (for longitudinal follow-up), offers the opportunity for early diagnosis, disease follow-up, and therapeutic monitoring. One example of this is plasma cfRNA profiling to discover disease-associated gene signatures. We conducted feasibility tests of the NanoString Low RNA Input assay using re-designed low input primers to analyze the expression profiles of plasma cfRNA.
Feasibility Tests and Results:
1. Primer efficiency assays by synthetic oligo standards:
Synthetic Ultramar DNA oligos to target 50 genes in the NanoString RNA panel were purchased from IDT DNA. The synthetic oligos were pooled and diluted in a series of dilutions to make the standards. Low RNA Input nCounter assays on the standards showed the lowest detection limit to be 30 molecules without false amplifications from non-template control. The average efficiency of the 50 primer pairs was 114% (range: 97%-136%) based on the standard curve analysis on the synthetic oligo standards.
2. Good correlation of cfRNA profiling between nCounter low input assay and real-time qPCR:
cfRNAs from 4 healthy donors were isolated from 1 ml cell-free plasma with the Norgen Plasma/Serum RNA Purification Kit. cfRNAs were amplified with the NanoString Low RNA Input kit using re-designed primers for 7 pre-selected genes from the NanoString Pancancer Immune Profiling panel (PCIP). PCR products were hybridized to the PCIP panel. The same primers were also used in the real-time qPCR analysis. Fold changes of 4 cfRNA samples to the universal reference RNA between nCounter and real-time qPCR data correlated well (R2 = 0.94) with good linearity (slope = 0.97).
3. Cell-free RNA amount increases with aging:
We further profiled cfRNA from 10 healthy individuals with 40 pre-selected genes from the PCIP panel to include abundant cfRNA genes found in the pilot study, oncogenes, and PBMC-derived genes. We found there is a trend of higher nCounter counts in the elderly population, which is consistent with a previous study.
1. cfRNA profiling can be performed on the NanoString nCounter platform utilizing optimized primers and 14-18 cycles of PCR amplification,
2. LOD of nCounter Low RNA Input assay is 30 copies using synthetic oligo standards.
3. Good correlation was seen between NanoString cfRNA data and real-time qPCR, one of the gold standards for cfRNA analysis.
4. cfRNA analysis of a 10-healthy individual cohort found the cfRNA amount positively correlated with age, suggesting elderly people tends to have higher circulating cell-free RNA in the blood.
5. The new improved protocol for low input RNA makes the NanoString nCounter platform a cost-effective and multiplexable molecular research tool for liquid biopsies.
Detection and Analysis of Free Circulating Long non-coding RNAs and mRNAs in Colorectal Cancer Pantients.
Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic
RNA is not confined to individual cells, but as was discovered in recent years, it is also part of different types of extracellular vesicles secreted by cells including ectosomes, exosomes, oncosomes and others.
Thus, RNAs isolated from these extracellular vesicles could be used as proxies to determine states of cells in various healthy or diseased tissues. While the focus is mainly on small circulating RNAs like miRNAs or piwi-RNAs, which are the most abundant species in these vesicles and outside of cells in general, we decided to focus on long circulating RNA species - long non-coding RNAs and mRNAs. While all RNA species allow analysis of their expression, long RNAs also allow detection of mutations, edits or splicing events. These might not only help to identify changes that are occurring during tumorogenesis but might also reveal mechanisms, that direct RNAs in these secretion vesicles.
In this work we analyzed publicly available sequencing data of exosomal RNAs1 as well as our data from extracellular vesicles derived from plasma of healthy volunteers and colorectal cancer patients. The analysis included presence of RNA regions, differential gene expression and splicing variants.
Overall, we detected changes in expression of some predicted splicing variants between healthy donors and colorectal cancer patients. Interestingly both publicly available and our own data contained a high levels of intronic sequences.
This work has been supported by the Charles University Research Fund (Progres Q39), by Charles University Research Centre program UNCE/MED/006 "University Center of Clinical and Experimental Liver Surgery" and by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic.
1 Li, S., Li, Y., Chen, B., Zhao, J., Yu, S., Tang, Y., Huang, S. (2018). exoRBase: a database of circRNA, lncRNA and mRNA in human blood exosomes. Nucleic Acids Research, 46(D1), D106–D112. https://doi.org/10.1093/nar/gkx891
Detecting Alzheimer’s Disease Risk Factors by qPCR Directly on Blood or Saliva Samples
TATAA Biocenter, Sweden
Apolipoprotein E4 (ApoE4) is a major genetic risk factor for sporadic or late onset Alzheimer’s disease, with two single nucleotide polymorphisms (SNP) rs429358 and rs7412 respectively, being major predictors to the development of the disease (1). Furthermore, Alzheimer’s diagnosed patients that carry a specific allele of the BDNF rs6265 single nucleotide polymorphism have been shown to lose memory and thinking skills more rapidly than non-carriers (2).
TATAA Biocenter has developed three robust SNP genotyping assays for identification of the ApoE and BDNF alleles that predict development of late onset Alzheimer’s disease. We demonstrate the accuracy of our assays through analysis of know genomic DNA standards, synthetic DNA as well as benchmarking against other commercial SNP genotyping assays.
In conjunction with our new TATAA Direct qPCR GrandMaster Mix, we show that the step of DNA extraction from blood or saliva samples can be entirely removed from the SNP genotyping workflow. Here we present supporting data for performing qPCR SNP genotyping directly on blood or saliva samples, without the need for DNA extraction.
(1) Rasmussen, Katrine Laura. "Plasma levels of apolipoprotein E, APOE genotype and risk of dementia and ischemic heart disease: a review." Atherosclerosis 255 (2016): 145-155.
(2) Boots, Elizabeth A., et al. "BDNF Val66Met predicts cognitive decline in the Wisconsin Registry for Alzheimer's Prevention." Neurology 88.22 (2017): 2098-2106.
Building High Quality NGS DNA Libraries from Ultra-small Fragments
LGC, Biosearch Technologies, United States of America
Researchers are extending the use of next generation DNA sequencing and library preparation kits to highly challenging sample types such as ancient DNA and FFPE purified DNA as well as very small chromatin immunoprecipitated (ChIP) samples and cell-free DNA.In this study we describe a modified, modern library prep method for the construction of high quality DNA fragment libraries from extremely challenging and small samples. Examples for the application of this technology include both synthetic libraries using various size dsDNA oligos (10 bp to 90 bp) to demonstrate protocol capabilities as well as experimental data with cell-free DNA and micrococcal nuclease-digested chromatin immunoprecipitated material.
Liquid Profiling of Head and Neck Cancer: Circulating cell-free DNA in Plasma and Saliva for minimally invasive Cancer Monitoring
1Department of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 2Department of Internal Medicine III - Hematooncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 3Department of Otolaryngology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 4Department of Oral and Maxillofacial Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 5Department of General Pathology and Pathologcial Anatomy, Technische Universität München, Munich, Germany; 6German Cancer Consortium (DKTK), partner site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany
In patients with head and neck squamous cell carcinoma (HNSCC), disease monitoring and detection of tumor recurrence are currently based on clinical examination, histological tissue analysis, and clinical imaging. This, however, does not sufficiently detect short-term changes in tumor burden. Tumor-derived materials, such as circulating nucleic acids, are present in body fluids of patients with cancer. The study of blood and saliva on molecular tumor constituents (liquid profiling) could enable better tumor monitoring and improve the early detection of recurrence. The aim of this work is, therefore, to analyze to which extent circulating, cell-free tumor DNA (ctDNA) and HPV DNA can be detected and which sample material (plasma vs. saliva) is more suitable for liquid-based tumor profiling of HNSCC patients.
In 91 patients diagnosed with HNSCC, blood was prospectively collected before therapy and in the course of the disease. In 9 patients, mutations in the primary tumor were analyzed with next-generation panel sequencing (45 genes) and ctDNA was examined in plasma with individually designed mutation-specific digital droplet PCR (ddPCR) assays. Further, all patients’ samples were analyzed by two mutation-specific ddPCR assays to detect commonly described hotspot mutations in HNSCC. This allows quantification of ctDNA even without prior tumor sequencing.
In 50 patients with tumors in the oropharynx and oral cavity, cell-free DNA (cfDNA) was tested for HPV (E7 oncogene-specific for HPV16) in plasma.
On average, 23 mutations (range 10 to 52) were detected in the primary tumor tissue by panel sequencing. In 25% (23 out of 91), tumor DNA was detected in plasma with mutation-specific ddPCR assays, in 22% already in early tumor stages (stage I (9%); stage II (13%)). In the course of disease monitoring an increase in tumor load (progressive disease) could be detected in the liquid biopsies on average 5,4 months (range 2 weeks to 13 months) earlier than by clinical manifestation.
In p16 positive patients (n=16) cell-free HPV type 16-DNA was identified in plasma in 39% (6 of 16) and thus allowed to identify the HPV type. In 50% of the cases, this was possible already at stage II. Specific mutations were observed in 75% of saliva samples (3 out of 4) and HPV16 DNA in 25% of cases (1 out of 4) to date.
Cell-free tumor DNA detection in plasma with digital droplet PCR is a promising monitoring tool for cancer profiling in early as well as advanced stages (III/IV) and could improve the early detection of recurrence. Saliva samples seem to be better suited for the detection of ctDNA than plasma. In HPV-associated tumors, circulating HPV DNA could be an additional profiling marker for disease monitoring of HPV-related cancer.
Anesthesia and Its Impact on miRNA Profiles in Circulating Extracellular Vesicles During Cancer Surgery
1Division of Animal Physiology and Immunology, Technical University of Munich, Freising, Germany; 2Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany; 3Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany; 4Department of General, Visceral, and Transplant Surgery, Ludwig-Maximilians-University Munich, Munich, Germany
While surgical resection represents the best chance of survival for patients with solid tumors, counterintuitive evidence suggests that surgery itself might facilitate tumor progression and metastasis. Data gathered in experimental and retrospective cohort studies indicate that anesthetic drugs commonly used in cancer surgery might have a differential impact on tumor cell biology, host immune response to surgery and tumor progression. The use of volatile anesthetic gases (VAG) may favor tumor cell growth and metastasis, while total intravenous anesthesia (TIVA) seems to exert anti‑tumor effects. Extracellular vesicles (EVs) and associated miRNAs are key mediators of intercellular signaling and metastasis, but little is known about how anesthetic drugs might impact circulating EVs.
To address this knowledge gap, we studied serum EVs in a cohort of colorectal cancer surgery patients. EVs were sampled prior to anesthesia using VAG (sevoflurane, n=9) or TIVA (propofol, n=8) and after tumor resection. EV‑associated miRNA profiles were analyzed by Next-Generation Sequencing (NGS) to identify transcripts with anesthesia-related perioperative changes in expression levels. Potential biological effects of miRNAs with altered pre- and post-surgical concentrations were assessed via in silico pathway analysis. Additionally, EVs sampled from patients in both groups and at both time points were biologically characterized.
In patients anesthetizes with both sevoflurane and propofol, pre- and post-operative miRNA profiles differed significantly. While there was significant overlap in the perioperative miRNA response, we detected five and 36 miRNAs that were specifically influenced by sevoflurane and propofol, respectively. Pathway analysis suggested that propofol-responsive miRNAs might exert inhibitory effects on cancer cell proliferation and migration and increase apoptosis, whereas miRNAs affected by sevoflurane did not impact these pathways. While size and marker protein composition of EVs were not altered by either type of anesthesia, EV concentrations in both groups were reduced after surgery.
In conclusion, our NGS data seem to match observations from retrospective cohort studies and point to EV-associated miRNAs playing a role in mediating the differential outcomes of cancer surgery using VAG or TIVA.
Sequence-specific release of EV-associated RNAs
Justus Liebig University of Giessen, Institute of Biochemistry, Germany
Within the last decade, extracellular vesicles (EVs) turned more and more into the focus, due to their important role in intercellular communication. Although it was known already that cells release vesicles to their environment during apoptosis (apoptotic bodies), also healthy cells massively release vesicles. These EVs are important factors for cell-cell communication, acting as vehicles of biological material between parental and recipient cells. EVs contain different classes of RNAs, such as mRNA, miRNAs and circRNAs. As shown for miRNA and circular RNAs, RNAs are selectively exported into vesicles [1-3]. However, the factors and mechanisms that contribute to this specificity remain elusive. For example, a so-called Exo-motif has been described for miRNAs, which, however, cannot be transferred to all RNA classes and for circRNAs a possible size-dependent export was suggested . In addition, only a few putative protein factors involved in packaging have been described .
To identify RNA sequence elements acting as export signals for the selective release of certain RNA species into EVs, we designed a modified in vivo SELEX approach (Systematic Evolution of Ligands by Exponential Enrichment). We generated a random sequence pool (N40), which was transfected and expressed in HEK293 cells. Specifically, several expression constructs were used, based on either an RNA polymerase II or III promoters. EVs were isolated from transfected cells, followed by RNA isolation, library preparation, RNA-seq analysis and bioinformatic identification of enriched RNA motifs. Sequence and motif enrichment analyses are now in progress. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs.
References:  Preußer et al. 2018, J Extracell Vesicles.;  Villarroya-Beltri et al. 2013, Nat Commun.,  Hinger et al. 2018, Cell Rep.
Analysis of Total RNA Yield and Purity Using Different Extracellular Vesicle Isolation Methods
1Sustainable Health, VITO (Flemish Institute for Technological Research), Mol, Belgium; 2Theoretical Physics, Hasselt University, Diepenbeek, Belgium; 3Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 4Laboratory for Soft Matter and Biophysics, University of Leuven, Leuven, Belgium
Minimally invasive liquid biopsies for diagnosis and monitoring of specific mutations in lung cancer struggle to achieve accurate results.1,2 An innovative approach is based on extracellular vesicles (EVs)—vesicles released in abundance by cancer cells in the bloodstream.3 RNA localized inside EVs is protected against degradation and, although highly fragmented, it contains mRNA fragments that reflect the mutational status of the parental cell.4 Therefore, EV-derived RNA (EV-RNA) has the potential to improve current liquid biopsy-based diagnostics in terms of sensitivity and accuracy. However, little is known about the effect of different EV isolation methods on the total vesicular RNA profile.5 In this study the EV-RNA yield and purity of three commonly used methods is compared: ultracentrifugation, size exclusion chromatography and a membrane affinity spin column-based method, each combined with the exoRNeasy kit for RNA extraction.
Up to fifty times more RNA is extracted from EVs with the spin column-based method compared to ultracentrifugation or size exclusion chromatography. The purity of RNA also differs greatly between the methods. After EV isolation by ultracentrifugation and size exclusion chromatography there is a clear fraction of ribosomal RNA whereas the spin column-based method co-isolates RNA complexes. These data show the need for a standardized method for RNA extraction from EVs for clinical diagnostic application to enable more reliable and sensitive mutation analysis.
1. Heitzer, E., Ulz, P. & Geigl, J. B. Circulating Tumor DNA as a Liquid Biopsy for Cancer. Clin. Chem. 61, 112–123 (2015).
2. Passiglia, F. et al. The diagnostic accuracy of circulating tumor DNA for the detection of EGFR-T790M mutation in NSCLC: a systematic review and meta-analysis. Sci. Rep. 8, 1–10 (2018).
3. El Andaloussi, S. et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).
4. Enderle, D. et al. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS. ONE 10(8): e0136133(2015).
5. Van Deun, J. et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J. Extracell. Vesicles 3: 24858 (2014).
Urinary Extracellular Vesicles: Unveiling the Most Appropriate Purification Method with a View to RNA Sequencing and Biomarker Profiling
1Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Freising, Germany; 2Department for Transfusion Medicine, Cell therapeutics and Haemostaseology, University Hospital LMU, Munich, Germany
Small extracellular vesicles (EVs) with a size ranging from 50 – 200 nm are released by cells into the extracellular space. By resembling both physiological and pathophysiological conditions of their parent cells, EVs are important mediators in intercellular communication. As such, they have already proven as putative biomarkers in various bodily fluids. Particularly urinary EVs, which could be sampled non-invasively, demonstrate enormous potential in early urogenital disease detection. Especially vesicle-enclosed microRNAs (miRNA), are in the focus of biomarker research based on their high stability and fundamental activity as post-transcriptional regulators. However, when using high-throughput profiling via small RNA sequencing, strong dependencies of the results on the chosen EV purification method have been already identified for blood samples, leading to low comparability and reproducibility. Thus, we aimed to identify the most appropriate urinary EV purification strategy and, moreover, to check for the congruency of obtained results.
Urine samples were collected from six healthy male volunteers in consistent compliance with the Declaration of Helsinki. After pre-clearing, five different urinary EV purification methods were applied based on different strategies: spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient. EV preparations were characterized by transmission electron microscopy. Purified EVs were further analyzed for size and concentration by fluorescence nanoparticle tracking analysis using urinary creatinine to normalize for individual differences. EV-specific and non-specific protein markers were assessed by Western blotting. Total RNA was isolated and quality controlled by capillary gel electrophoresis prior to small RNA library preparation. Generated cDNA was fractionated by gel electrophoresis for miRNA size selection. Small RNA sequencing was performed using HiSeq 2500 (Illumina). Generated reads were processed according to an in-house developed pipeline and further analyzed by the DESeq2 algorithm.
All applied urinary EV purification methods yielded enough EVs and RNA for subsequent small RNA sequencing, albeit significant differences could be observed. Moreover, Western blot analysis revealed substantial co-purification of uromodulin in some EV preparations going hand in hand with higher sequencing readcounts. Exploratory data analysis of DESeq2-normalized reads further pointed towards purification-dependent differences in miRNA profiles, emphasizing the need for the combined evaluation of biochemical properties, proteomics and transcriptomics.
Summed up, care should be taken when selecting urinary EV purification methods appropriate for downstream analyses and the initial research question. Moreover, proper EV characterization is indispensable to ensure comparability and reproducibility of generated results.
Validating Sensitive Workflows To Analyze RNAs in Tumor-derived Extracellular Vesicles in Serum and Urine Samples of NSCLC Patients
1Bayer AG, Germany; 2Qiagen, Germany; 3Department of Tumor Biology, Germany
Liquid biopsy is defined as molecular analysis of rare cells or cell-free nucleic acids circulating in blood or in other biofluids. The concept of liquid biopsy aims at closely monitoring the status of a disease or treatment efficacy in a simple, fast, cost efficient way and at any point in time with minimal risk and burden for the patient. A preferable approach to investigate “liquid” RNAs is to focus on circulating extracellular vesicle (EV)-cargo (like mRNA and miRNA) rather than on cell-free RNAs, as the former is well protected from RNase degradation and therefore, more stable. However, the volumes of available blood samples can be limiting for molecular analysis and “liquid” RNAs are normally present in low abundance. Therefore, detection and quantification of RNAs in the blood requires robust and highly sensitive technologies.
In this pilot study, we investigated the absolute expression of 10 EV-transcripts typically involved in cancer development and chemotherapy resistance in longitudinal serum samples of 12 non-small cell lung cancer (NSCLC) patients (age range 37-76 years) treated with cisplatin at the Department of Inner Medicine II, University of Ulm, between May 2011 and August 2012.
Blood samples were collected at baseline, after 3-6 months and after 9-12 months of chemotherapy from patients showing objective clinical response. EV-RNAs were isolated by sequentially processing obtained serum samples with miRCURY® exosome kit and miRNeasy kit (both Qiagen, Hilden, Germany). Afterwards, the status of selected EV-transcripts was investigated via ddPCR (Biorad, Milan, Italy). Furthermore, we compared commercially available kits for the extraction of EVmiRNA from serum (exoRNeasy S/P Midi Kit) and urine (miRCURY Exosome Cell/Urine/CSF Kit) as well as cfmiRNA from serum or urine (miRNeasy S/P advanced). Extraction technologies were compared by measuring miRNA quantity and recovery of the exogenous control cel-miR-39.
After start of chemotherapy, we observed a downregulation of at least 2-fold of the following potentially cancer-related EV-transcripts : PTEN, ERBB2, FOSL1, IL-8, MET, RPS27A, SF3B1 and of the following housekeeping EV-mRNAs: ACTB, HIST1H3H and HSPA1A, in all patients. In addition to EV-transcripts, we showed an increased concentration of EVmiRNAs in serum and urine of NSCLC patients. However, using cel-miR-39 as spiked-in control, high extraction variabilities in patient samples were shown.
In this project, we demonstrated dynamic changes of cancer-related EV-mRNA expression in serum samples of NSCLC patients during cisplatin treatment. In addition to EV-transcripts, EVmiRNAs in serum of NSCLC patients might be useful as valuable “liquid” RNA biomarker as well.
This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.
Serum-Free Media Supplements Carry MiRNAs That Co-Purify With Extracellular Vesicles
Johannes Gutenberg University of Mainz, Germany
Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing fetal bovine serum (FBS) or a defined media supplement as nutrient. Recently, Wei et al. (2016, PMID: 27503761) reported that miRNAs are co-isolating with EVs when harvested in media supplemented with FBS or vesicle-depleted FBS. To avoid serum-associated miRNA contamination, we performed RNA-Seq of EV-associated miRNAs derived from primary oligodendrocytes cultured under serum-free conditions and subsequently validated miRNAs by RT-qPCR including media and supplement controls. Intriguingly, several EV-associated miRNAs were robustly detected in un-conditioned media subjected to the EV-isolation protocol and the media supplements NS21 and B27, which are routinely used for neural cell culture. RNAse and detergent treatment of NS21 removed most but not all of the contaminating miRNAs. Detailed analysis of NS21-supplement by screening individual components excluded BSA as major source of the miRNA contamination and identified a single component as carrier of miRNAs. Media supplement lacking the miRNA-carrying components however appears to affect cell viability, indicating that deprival of this specific supplement is not a solution to get rid of contaminating miRNAs. Based on these findings, we designed a new RNA-Seq strategy including EV-samples collected under conditions of supplementation or supplement-deprivation as well as supplement only control.
Taken together, our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV-samples. Consequently, analysis of EV-RNA needs refined strategies including respective media controls.
Systematic Comparison of Extracellular Vesicles from Human Arterial and Venous Blood: Highly Identical microRNA Expression Indicates Equal Use for Biomarker Applications
1Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany; 2Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Germany; 3IMGM Laboratories GmbH, Planegg, Germany; 4Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Germany
Extracellular vesicles (EVs) are crucial vehicles for intercellular communication in cardiovascular disorders such as cardiac hypertrophy and cardiac remodeling. To increase analytical specificity, biomarker studies often favor EV-associated microRNAs (miRNAs). While blood for diagnostic or research purposes is usually sampled using peripheral venous access, arterial lines inserted for monitoring are more convenient and often preferred in critically ill patients undergoing intensive care unit therapy or major surgery. This hampers comparisons between different pathologies and studies. We therefore tested whether EV-associated miRNA profiles depend on sample site location. Our study population included 20 cardiac surgical patients in which blood was simultaneously drawn from the radial artery and from the internal jugular vein prior to surgery. EVs were isolated using a polymer-based precipitation method, and total RNA was subsequently extracted. We characterized EVs by Nanoparticle Tracking Analysis, Western blot analysis and transmission electron microscopy. After small RNA sequencing, differential expression analysis was performed (DESeq2). Applying stringent filtering criteria to our data, no significantly regulated miRNAs were detected. Using less stringent filtering, we detected a subset of four miRNAs (miR-223-3p, miR-379-5p, miR-493-5p, miR-542-3p) slightly upregulated in arterial samples. We quantified these miRNAs by RT-qPCR. However, only one transcript (miR-493-5p), showing the highest regulation (1.6-fold), could be validated. Both arterial and venous EVs had spherical morphologies and equal particle concentrations in serum. However, arterial EVs were marginally smaller. Common EV protein markers were detected independent of sample type. Our data indicate that arterial vs. venous blood sampling should not represent a likely confounder when studying differentially expressed EV miRNAs. Therefore, data from studies using arterial or venous EV samples are probably comparable, with the exception of specific pathologies (e.g. severe pulmonary disorders).
Grp78 Plays A Crucial Role In The Exosome-Promoted Survival Of Irradiated Squamous Head And Neck Cancer Cells
Helmholtz Zentrum München, Germany
Background: Exosomes are nanometer-sized extracellular vesicles with functions in intercellular communication. Recent findings demonstrate that exosomes derived from irradiated HNSCC cells confer a more radioresistant phenotype to recipient cancer cells*. Here, we investigate the mechanism how exosomes promote survival in the radiation response of the head and neck cancer cell line BHY.
Methods & Results: Exosomes were isolated from conditioned medium of irradiated and non-irradiated cells by serial ultracentrifugation. Increased Grp78 was identified in exosomes from irradiated donor cells by whole proteome analysis** and by western blot. Moreover, we detected the localization of Grp78 at the exosome surface. In accordance to the increased Grp78 content of exosomes we also observed increased Grp78 expression in exosome donor cells (predominantly at the cell surface) after irradiation, while mRNA levels remained constant. We further observed increased surface expression of Grp78 in two other HNSCC cell lines; CAL-33 and FaDu. To test whether the exosomal Grp78 content can influence the Grp78 content of exosome recipient cells we cultured BHY cells with exosomes from irradiated and non-irradiated cells. We find enhanced Grp78 levels after incubation with exosomes from irradiated cells. To check a potential role of Grp78 in cell survival after irradiation we overexpressed the gene in BHY cells and performed a clonogenic survival experiment. Here we found that Grp78 alone enhances the radioresistant phenotype of BHY cells.
Discussion & Conclusion: Our results suggest a pivotal role of Grp78 transferred by exosomes in the radiation response of BHY cells. Radiation increases the cellular and exosomal Grp78 amount and moreover vesicle-mediated communication leads to enhanced Grp78 levels in exosome recipient cells. As Grp78 supports radioresistance, the transfer of this protein may contribute to the observed prosurvival effect of exosomes from irradiated donor cells. This study provides new mechanistic insights into the effects of exosomes in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy.
* Mutschelknaus L, Peters C, Winkler K, Yentrapalli R, Heider T, Atkinson MJ, Moertl S. Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation.PLoS One. 2016 Mar 23;11(3)
** Mutschelknaus L, Azimzadeh O, Heider T, Winkler K, Vetter M, Kell R, Tapio S, Merl-Pham J, Huber SM, Edalat L, Radulović V, Anastasov N, Atkinson MJ, Moertl S. Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells. Sci Rep. 2017 Sep 29;7(1):12423.
Analysis Of DNA Content From Human Melanoma Cellline Derived Extracellular Vesicles
1Biotechnology in Animal Production, IFA Tulln, Austria; 2Institute of Animal Breeding and Genetics, Vetmeduni Vienna; 3Research Group Oncology, Equine Clinic, Vetmeduni Vienna; 4Institute of Pathology and Forensic Medicine, Vetmeduni Vienna; 5Department of Dermatology and Venereology, Karl Landsteiner University of Health Sciences
Extracellular vesicles (EVs) are a heterogenous population of membrane surrounded structures released by cells with varying cargo including nucleic acids, proteins and lipids. It is now established that these EVs have a significant role in cancer signaling. The nucleic acid (NA) content of EVs varies in amount and is both covering the nuclear (nDNA) and mitochondrial (mtDNA) genome. EVs are routinely extracted by differential centrifugation of cell culture supernatant, thereby separating enriched EV fractions (apoptotic bodies; microvesicles; exosomes). In this study, we characterized these fractions in three human melanoma cell lines; human fibroblasts and human melanocytes as controls. The EV-cargo was analyzed regarding their mtDNA: nDNA ratio. Furthermore, by using Proteinase and DNase treatments the surface-bound nucleic acids were digested to estimate the Distribution of DNA on the vesicular surface versus intravesicular DNA.
Melanoma Cell lines (A375, SKMel28, 518A2), human melanocytes and human fibroblasts were cultured in exosome depleted media for 48hrs; EVs were isolated with differential centrifugation of cell culture supernatant. A digestion step was performed using Proteinase K and Dnase followed by DNA extraction of each respective pellet enriched for apoptotic bodies, microvesicles and exosomes. MtDNA to nDNA ratio was quantified by qPCR (nuclear DNA: single copy gene MIA GenBank [NC_000019.10], multi-copy gene NPIPL (Sudmant, Kitzman et al. 2010) [NC_000016.10]; and mitochondrial DNA: MT-ND4 GenBank [NC_012920]).
Preliminary data indicates that the ratio of nDNA to mtDNA increases with each step in differential centrifugation. This suggests that in exosomes the relative amount of mtDNA is reduced compared to microvesicles, and even more in apoptotic bodies, irrespective of analyzed cellular origin. In an ongoing study the ratio of extravesicular vs intravesicular nucleic acids in the respective centrifugation pellets is analyzed.
The ratio of nDNA to mtDNA increases with each step in differential centrifugation, both in melanoma and fibroblast cell lines. Further research is needed to discriminate further between extravesicular vs intravesicular nucleic acids in the respective centrifugation pellets, and to elucidate the underlying mechanisms of nucleic acid distribution in EVs. This knowledge will be further transferred to the clinical level where tumour-derived EVs isolated from blood and the corresponding nucleic acids are going to be analyzed.
This work was supported by grant LS15-020 (to J.P.B.; F.T and S.B.) from Life Science Calls, Niederösterreichische Forschungs-und Bildungsgesellschaft (NFB; http://www.nfb.at/).
P3py: A Python Module For The Multi-array MicroRNA Detection Data Analysis And The simulation Of Biomarker Diffusion And Reaction
BTU Cottbus Senftenberg, Germany
MicroRNAs are small, conserved, noncoding RNAs regulating gene expression at the post-translational level, altered microRNA profiles have been implicated in many human diseases and thus used as clinical biomarkers. We developed a diffusion driven microbead multiple assay and combined it with an antibody based miRNA detection. The P3py python package was developed for the multiarray data analysis. P3py consists of two parts: microarray experimental data analysis and the computational simulation of microRNA diffusion and reaction. For the experimental data analysis, P3py can be used to preprocess microarray raw data, visualize fluorescence signal intensity using the 2D-space contour plot or heat map, statistical tests for the microbead distribution against the Complete Spatial Randomness (CSR), dose-response data curve fitting over time, fitted curve comparison under different experimental conditions. For the computational simulation, P3py solved the microRNA diffusion and reaction equations, and tried to predict the hybridized target concentration over time, there is a high correlation between the predicted and experimental hybrid target concentration.
Phenovault: An Open-Access Resource For Analysing RNAi And CRISPR Screens
siTOOLs Biotech, Germany
RNAi and CRISPR screens give rise to a wealth of information on gene function. Recently published analysis focuses on a few top-scoring genes. Consequently, complete genome-scale screening data often lies buried within supplementary materials, invisible and untapped. The Phenovault is a growing database and analysis suite hosted by siTOOLs Biotech that contains complete, reagent sequence/ID-linked datasets from published RNAi/CRISPR screens. With over 20 million data-points, the Phenovault is the largest curated RNAi screening repository. Together with public and proprietary algorithms that harness the dominant microRNA seed-based behaviour of siRNAs, the Phenovault helps researchers uncover novel insights into their 1) siRNA reagents, 2) target genes and 3) RNAi screening datasets.
Optimal Use Of Statistical Methods To Validate Reference Gene Stability In Longitudinal Studies
1Université Paris Descartes, France; 2INSERM U1124, Myelination and Nervous system pathologies - Group
Multiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Indeed, as their respective approaches to calculating reference gene stability is different, their suitability has to be tested for a given experimental setting. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation analysis and Pairwise ΔCt method) in a longitudinal setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise ΔCt were found to be ill suited due to a fundamental assumption in their stability calculations. Whereas, NormFinder and Coefficient of Variation analysis fare better provided they are used complementarily. We therefore devised a workflow combining these two methods for validating reference genes in developmental studies. This workflow proves to be more robust than any of the methods used individually.
A New Method to prepare DNA Libraries for NGS by using SD DNA Polymerase
Fragmentation of DNA is the first and very important step in preparing nucleic acids for NGS. Here we report a new method of NGS library construction based on a novel Fragmentation Through Polymerization (FTP) technique, which is simple, fast and user-friendly method. It generates double-stranded DNA fragments that are suitable for the direct use in NGS library construction, and allows eliminating the need of an additional step of reparation of DNA ends. The method allows simple and efficient addition of adaptors to the DNA fragments.
The FTP technique presumes the introduction of random nicks into the double stranded DNA by nuclease with simultaneous filling-in the DNA sticky ends which appeared as the result of nuclease action. Filling-in of the sticky ends requires strand displacement activity which is provided by Bioron’s SD polymerase, unique thermostable enzyme with strand displacement activity. The same enzyme can be used to amplify DNA fragments as soon as adapters are ligated to the ends of the fragment obtained.
In total, the procedure can be completed in 2-3 hours starting from genomic DNA and resulting in excess of PCR-fragments with adapters suitable for Illumina (or IonTorrent) sequencing.
BIOCEV GeneCore – More Than Just Gene Expression Profiling
Institute of Biotechnology CAS, Czech Republic
We present services of BIOCEV GeneCore - the best equipped core facility and service provider in the field of gene expression in Central Europe. We have broad experience in quality control (QC e.g. Fragment Analyser) in a single cell analysis (automated cell picking ALS Cellcelector), high-throughput and digital PCR (Fluidigm Biomark, BioRad QX200 Droplet Digital PCR System) and NGS library preparation.
We emphasise quality control, which is often neglected. Effective QC is based on the use of molecular tools to control contamination (RNA/DNA spikes), genomic background (ValidPrime) and quality of RNA (ΔAmp, RIN). We also take part in development of these methods to facilitate analysis of gene expression starting from bulk samples, down to the level of individual cells (direct lysis).
In addition to conventional qPCR analysis, we focus on single cells expression profiling and multi-analyte approach. Analysis of DNA/RNA/protein in parallel in one sample even on the single cell level provides comprehensive tool to map gene expression, to characterize types of cells, to determine the degree of differentiation and to study the pathological condition.
Currently, we provide assistance with library preparations and experimental design of RNA-Seq experiments, which are key preconditions for a successful project. Moreover, we offer new Two-Tailed PCR for ultrasensitive analysis of microRNAs based on an innovative novel design with a RT primer sensing the microRNA using two connected hemi-probes exceeding sensitivity and superior specificity is achieved.
MLH1 Promoter Hypermethylation: Development and Validation of a Methylation-Sensitive High-Resolution Melting (MS-HRM) Assay for use in a Lynch Syndrome Pre-Screen Pathway
1Sarah Cannon Molecular Diagnostics, HCA Healthcare UK, London, UK; 2Faculty of Medicine, Imperial College London, London, UK; 3Department of Histopathology, UCLH NHS Foundation Trust, London, UK; 4MethylDetect ApS, Aalborg, Denmark; 5Pomeranian Medical University, Szczecin, Poland
Lynch Syndrome (LS) is associated with germline mutations in genes encoding the mismatch repair proteins, leading to mismatch repair deficiency (dMMR). dMMR can also arise somatically by promoter hypermethylation-mediated silencing of the MLH1gene. In colorectal cancer, there is a strong association between the BRAFVal600Glumutation and MLH1promoter hypermethylation, thus analysis of these markers, along with microsatellite instability (MSI), constitute components of the NICE-mandated LS pre-screen pathway. In the absence of a CE-IVD solution, we sought to develop and validate an MS-HRM based, MLH1promoter methylation assay for clinical use, using primers and control material from MethylDetect ApS, and bisulphite conversion and HRM-PCR mastermix kits from Qiagen.
Paired tumour/normal DNA was extracted from 20 FFPE samples: 12 colorectal, 4 prostate, 2 endometrial, 1 bladder, 1 unknown. Ten samples were MSI-High (MSI-H), 10 MSI-Stable (MSS); 5 were known dMMR, 2 MMR-normal; 4 were known BRAFVal600Glu, 10 BRAFWT; 1 from known LS, 3 from suspected LS. 20μL (5ng/μL) DNA was bisulphite converted (bsDNA) and 2-3μL used per HRM reaction. These were run on a Qiagen Rotor-Gene Q with 2-4 technical replicates for 45-50 cycles, with a melt start of 65-69oC and finish of 83-95oC. The paired-normal sample was used as the unmethylated baseline on a per-sample basis.
Repeated optimisation runs showed that 3μL bsDNA input, with 4 technical replicates, x50 cycles, and 69-83oC melt were the optimal parameters. All 11 MSS samples scored as unmethylated, as did the 4 known and suspected LS samples. The remaining 6 MSI-H samples (including 3/4 BRAFVal600Glu) scored as hypermethylated. MethylDetect control samples showed a limit-of-detection of 1% methylation.
All validation samples scored as expected, except for one BRAFVal600Glusample that scored unmethylated; this however came from a suspected LS patient and thus warrants further investigation. This assay proved to be rapid, cost-effective, sensitive, and specific.
Detection And Quantification Of MicroRNAs Using A Multiplex Microbead Assay To Accompany Quantitative PCR And Digital PCR Methods
1BTU Cottbus-Senftenberg, Chair of Multiparameter Diagnostics, Senftenberg, Germany; 2GA Generic Assays GmbH, Blankenfelde-Mahlow, Germany; 3University of Potsdam, Institute of Biochemistry and Biology, Professorship of Immunotechnology, Potsdam-Golm, Germany
MicroRNAs (miRNAs) are short (~ 22 nt) RNA molecules which act as post-transcriptional regulators on protein expression, cell differentiation, cell communication and immune defense. They can circulate into the bloodstream and other body fluids. MiRNAs are important clinical biomarkers for diseases, such as cancer, immune-related or cardiovascular diseases. Specific miRNA profiles e.g. from serum or plasma are promising tools for early in vitro diagnostics. Therefore, there is a high interest to develop sensitive, specific and robust methods for miRNA analysis. Quantitative PCR (qPCR), digital PCR (dPCR), microbead (MB) assays, microarrays and NGS are commonly used for the detection and quantification of miRNAs.
Our focus is the development of a non-invasive, multiplex MB-based detection system to accompany (quality control) the determination of specific circulating miRNA profiles from the serum of cardiomyopathy patients by qPCR and dPCR. An important aspect is to design the system in such a way that no amplification reaction is necessary.
Size/dye encoded MB were coupled via biotin-neutravidin bond with DNA capture probes which specifically targets the miRNAs of interest (miR-21, miR-197, miR-379, Let-7f). To achieve a direct comparability of the MB- and PCR-based methods, the miRNAs were first polyadenylated, afterwards hybridized to the capture probe loaded MB and detected via an ATTO 647N-labeled poly(T) sequence by using our in-house multispectral fluorescence imaging platform , which is based on a fully automated multispectral fluorescence microscope. For qPCR and dPCR, the polyadenylated miRNA was transcribed into cDNA and used in the same dilutions as in the MB assay.
Results and Conclusion
We developed a MB assay which enables the multiplex detection of so far four miRNAs from one sample in a concentration-dependent manner down to the lower nanomolar range. The comparison of the detection limit of the current MB assay with the quantitative PCR methods shows that these have at least 100 times higher sensitivity. The MB based miRNA detection is currently no more sensitive than the qPCR or dPCR. However, it offers some advantages regarding the multiplex degree as well as the amplification-free and isothermal reaction. Multiparametric analyses are relevant because they can work more cost-efficiently, require less sample material and more parameters can map disease patterns more accurately and thus make patient-related statements possible. Our assay has no amplification bias, as PCR based methods, since amplification can lead to a shift in certain target molecules and may serve as tool for miRNA sample quality control.
To increase the sensitivity of MB-based miRNA detection, we are working on the generation of poly(A)/(T) hybrid antibodies as well as miRNA-specific antibodies and the implementation of the assay into a microfluidic platform.
 Rödiger et al. (2013) Adv. Biochem. Eng. Biotechnol. DOI: 10.1007/10_2011_132
Developing a Customizable Panel of Real-time qPCR Assays on a Microfluidic Device for Respiratory Tract Pathogen Detection
Thermo Fisher Scientific, United Kingdom
Respiratory tract infection is the most common infection in the world, presenting serious health issues to individuals and communities. A wide variety of bacteria and viruses are capable of causing upper and lower respiratory tract infections. Panel based testing using molecular methods to identify these pathogens has clear advantages over non-molecular or single target molecular testing.
A novel set of TaqMan® qPCR assays was developed to over 40 distinct respiratory pathogen targets, including bacteria, DNA viruses, RNA viruses, and fungi. These FAM™ dye-labeled assays were pre-loaded and lyophilized onto wells on TaqMan® Array Card™ (TAC), a microfluidic device that enables 8 samples tested for up to 48 targets simultaneously.
We report test data for sensitivity, specificity, accuracy and reproducibility of these respiratory tract pathogen assays on the microfluidic TAC.
The application enables researchers to study a large number of respiratory pathogens in a single reaction with a simple workflow, fast turnaround time, and high throughput yet flexible sample/target combinations.
Methods to determine Limit of Detection and Limit of Quantification of a quantitative real-time PCR (qPCR).
Multid Analyses AB, Sweden
Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.
A Winter`s Week Tale in Sheffield Virology Department
Sheffield Teaching Hospitals NHS Trust, United Kingdom
Sheffield Teaching Hospitals NHS Foundation Trust (STH) is one of the UK’s biggest and most successful providers of hospital and community based healthcare. We provide a comprehensive range of local services to the residents of Sheffield, Yorkshire and Derbyshire. The virology department contains Serology, Molecular and Development sections employing 28 members of staff. We perform NA extractions in the Category 3 laboratory using the MagNaPure 96 or Qiacube extraction systems. We have dedicated rooms for each PCR stage for a unidirectional workflow. Weekly plan to cover all in PCR assays within stated turnaround times requires precise planning. ABI7500, Rotorgene Q, Panther, Veris and LC480 platforms to perform testing of Respiratory viruses, Enterovirus, Measles, Mumps, Norovirus, HSV, VZV, Atypical Pneumonia screen, MERS, P.jiroveci, BKV, EBV, HHV6, Parvovirus, CMV, HIV, HBV, HCV, CT, GC and T.vaginalis using qualitative or quantitative real time PCR. Our respiratory viruses’ panel contains 16 viruses covering Influenza, Parainfluenza, Respiratory Syncytial Virus, Human Metapneumo Virus, Corona virus, Rhinovirus and Adenovirus with DNA and RNA internal controls. During the Winter season there is a substantial increase in the number of respiratory samples tested. Commercial assays as well as in house developed assays are used for testing. All our in house assays have the same amplification profile enabling us to perform different PCR tests together and providing rapid results of urgent tests. Plasmid based positive controls as well as strict in house PCR batch approval systems ensure a consistent and efficient diagnostic procedure.
Quicker results can be obtained by:
- Using Multiplex PCR. Our Multiplex Respiratory viruses’ PCR panel requires 6 wells for screening.
- Increasing plate size for testing. Using 384 well plates instead of 96 wells plates has increase our capacity 4 fold.
- Information technology packages such as Roche cITM or result transfer option from ABI7500 system help greatly for efficient, accurate and timely result processing.
- Automation such as pipetting stations eliminate any pipetting errors and reduce hands on time .
Master mixes play a very important role for the development of and testing of PCR reactions. Highly stable and efficacious master mix will help producing consistent and correct results even if sample viral load is very low. The PerfeCTa qPCR tough mix and QScript custom XLT 1-step RT-qPCR toughmix are used for our in house PCR assays. Development of the assays was based on Taqman technology using the ABI7500 system. However, we are now in the process of revalidating our in house assays on the Roche Flow system using 384 well plates. I have chosen the week starting 7th January 2019 to highlight winter pressure on our Virology department and how we tried to overcome most of these issues. When compared to a week in summer there was a 4 fold increase in the respiratory samples received.
Effects Of The Use Of Degenerate Bases In Primers On Quantitative PCR Measurements
In our institute, we develop dual research programs to respond both to the armed forces aims and civilian uses. Different experiments are thus conducted in the field of NBC risks. To this aim, we studied the effects of degenerate bases incorporation on quantitative PCR measurements to improve diagnostic of biological agents presenting nucleotide polymorphism. Primers were thus modified using either inosine or 5-nitroindole, two “universal” bases. Different parameters were evaluated such as the type and number of substitutions, their location and their contiguity effects. Asymmetric PCR were also conducted in order to verify substitution’s effect on PCR stability. First, we designed forward and reverse primers containing 1 to 3 substitutions at different location of the primers. Then, we studied the effects of 6 substitutions placed on contiguity or not and on the first position of the 5’ end of the primer or not. All these primers were tested on synthetic matrices in term of PCR amplification efficiency and specificity of the reaction. For this, delta Ct difference and variation in melting temperature were calculated using amplification occurring without modified primers as control.
Our work revealed that nitroindole substitution led to a decrease in PCR performance whatever number of substitution on the 5’ or middle of the reverse primer. When one to three nitroindole were placed on the 3’ end of the primer, no amplification occurred.
When using inosine substitutions, we demonstrated that a single residue placed in the 3’ end of the primer had a negative effect on PCR performance. Such effect was potentiated with the increase in number of substitutions. When 6 inosine substitutions were present, inosine residue on the first 5’ end of the primer appeared essential. Moreover, PCR amplification was less destabilized when groups of 2 consecutive inosine were present on the primers.
Finally, this study demonstrated that, in our model, a better amplification occurred when residues were substituted by inosine instead of nitroindole. We also showed that bases modifications by inosine on the 3’ end of primer strongly destabilized PCR. Moreover, in case of multiple substitutions by inosine, modification in the first 5’ end of primer associated with contiguous degenerated residues clearly improved amplification. Further investigation will be done to confirm these effects.
Evaluation of DNA Fragmentation Methods for Implementation of cgMLST in Routine Analysis
Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany
NGS currently represents the highest-resolution method for strain typing. In combination with a suitable analysis of the data, such as the comparison of the core genome multilocus sequence typing (cgMLST), NGS has the potential to differentiate the bacterial isolates of a species much more precisely than with the typing methods previously used in routine. Potential sources of infection can thus be identified more quickly. The use of NGS technology in routine analysis, as part of the official monitoring of food, requires an optimization of the corresponding workflow with regard to sequencing quality and time/cost ratio.
In addition to the decision whether to generate DNA libraries using PCR-free or PCR-dependent methods, the choice of the DNA fragmentation method is a crucial factor. Besides enzymatic methods, physical DNA fragmentation using ultrasonic is also possible. A key factor here is the generation of overlapping partial sequences (reads) that can be combined into coherent sequences after sequencing. The average number of reads per nucleotide position in the genome is called coverage and is a parameter for the quality and precision of the assembled sequence. The prerequisite for high coverage is the generation of strand breaks as randomly and uniformly as possible. Coverage can vary depending on the type of fragmentation method selected and the DNA sequence of the organism. Fragmentation, particularly in enzymatic processes, is not always completely random, but can occur with varying frequency in specific DNA sequences. Possibly, DNA libraries are created with an undesired wide size distribution with fragment sizes that deviate significantly from the desired length. Fragments that are too short or too long are removed during purification steps or size selection, which reduces the coverage of the affected DNA sequences or might lead to the fact that no sequence information at all is obtained for some DNA regions. Due to the short reading length during NGS sequencing, only parts of long DNA fragments are sequenced, which means that a lot of sequence information is lost.
In order to evaluate the susceptibility of different methods to the sequence-induced generation of irregular fragment sizes, their application to bacterial DNA with different GC contents (Campylobacter coli with ~30 % GC content and Salmonella enterica with ~50 % GC content) was tested in triplicate and evaluated in terms of time, cost and size distribution. Fragmentation was performed physically by ultrasonic on the one hand and enzymatically by tagmentation or two alternative enzymatic methods on the other hand. The generated fragments were separated by capillary electrophoresis and the size distributions of the individual isolates were compared before and after size selection. When enzymatic kits were used, the DNA could be overfragmented due to sequence-specific fragmentation, while physical fragmentation is easier to standardize due to less fragmentation bias.
FASTFISH-ID™: ThermaGenix’s Rapid DNA Authentication of Any Species of Commercial Fish Using Bio-Molecular Systems’ MIC sPCR Cycler.
1Thermagenix, Inc,, Natick, Massachusetts, United States of America; 2Institute for Global Food Security, Queen’s University Belfast, Belfast, United Kingdom; 3School of Environment and Life Sciences, University of Salford, Salford, United Kingdom; 4Bio Molecular Systems, Upper Coomera, QLD, Australia
PROBLEM: Species mislabeling/substitution in commercial fish products threatens the economic welfare, product safety, and sustainability of the seafood industry. The sequence of the mtCO1 DNA barcode region serves as a universal molecular tag for species identification. COI DNA barcoding is not suitable for routine species monitoring along fish supply chains due to slow and costly off-site sequencing. SOLUTION: ThermaGenix’sFASTFISH-ID™ is a revolutionary low-cost DNA test that empowers anyone anywhere along the supply chain to rapidly authenticate ANY species of commercial fish without sequencing or having to do separate tests for each species. FASTFISH-ID™ provides answers in just two hours or less using a simple “sample-in, answer-out” test implemented in the MIC qPCR Cycler, a highly accurate, portable and affordable instrument from Bio Molecular Systems. METHOD: FASTFISH-ID™ converts species-specific COI DNA barcode sequences into highly-reliable fluorescent signatures that are then compared to a cloud-based reference library for immediate answers. Most importantly, FASTFISH-ID™ integrates seamlessly into existing regulatory DNA barcoding protocols: If needed, mislabeled and unknown samples rapidly identified with FASTFISH-ID™ can be directly sequenced for regulatory compliance or for reference library addition, respectively. Sample preparation is also fast: samples are just disrupted in a proprietary reagent and then diluted into the test for immediate identification. RESULTS: FASTFISH-ID™ was validated by two independent laboratories in England and Northern Ireland using a blinded panel of 18 commercial fish species comprised of 1-8 biological replicates (three technical replicates each) with 96%-100% success. FASTFISH-ID™ was also successfully tested on a research vessel in the North Atlantic. SIGNIFICANCE: FASTFISH-ID™’s turnkey “sample-in, answer-out” operation, portability, and cloud-based analysis software makes authentication of any fish species easier, faster, and cost-effective. Rapid and convenient FASTFISH-ID™ species authentication will protect the seafood industry against the vulnerabilities of species substitution/mislabeling. The same technology can be used for analysis of virtually any group of animals, plants, or microbes on Earth by designing the appropriate PCR primers and probe sets covering numerous genera and species.
Is The High-Throughput human OpenArray® System Useful For Profiling miRNAs In Melanoma Regression In A Swine Model?
1INRA, France; 2CEA, France
Cutaneous Melanoma (CM) is the most aggressive cancer of the skin due to its high metastatic potential. Despite recent encouraging results from target therapies, treatment of invasive melanoma is still challenging and new data on melanoma biology are required. MicroRNA (miRNA) are post-transcriptional regulators of gene expression. Altered miRNA expression profiles and the identification of their targets in cancer imply the high potential of miRNA as diagnostic and prognostic markers for development of new therapeutic strategies. The Melanoma-bearing Libechov Minipig (MeLiM) model is a relevant animal model of CM since melanocytic lesions are similar to human counterparts. MeLiM pigs are born with CM that progress to spontaneous regression leading to a high rate of survival even in metastasis-bearing animals. In this study, we investigated whether miRNA could be involved in this fascinating process.
Even if the release of the pig genome sequence in 2012 was a major step for using pig models for biomedical research, some data are always limited in swine. For example, in miRbase 21, only 382 sequences from Sus scrofa miRNA precursors are listed against 1881 for Homo sapiens. For this screening, we tested the use of TaqMan® OpenArray® human MicroRNA Panels (Pool A and B, ThermoFisher) charged with 750 human miRNA to compare quickly and easily, miRNA profiles between progressive and regressive MeLiM tumors. With 60% of positive amplification, results show that this approach could be an alternative of miRNA sequencing for miRNA screening in some species where “large” dedicated tools are missing.
New Advances in Two-Tailed RT-qPCR
1Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic; 2Faculty of Science, Charles University, Prague, Czech Republic; 3Faculty of Science, Palacký University, Olomouc, Czech Republic; 4TATAA Biocenter, Gothenburg, Sweden
MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as clinical biomarkers. In 2017, we presented a highly specific, sensitive and cost-effective method to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to termini of the targeted miRNA, connected by a hairpin structure. Here, we summarize the applications and advances in Two-tailed RT-qPCR in last two years. We used the two-tailed technology to create a panel for quality control (QC), monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The application of the QC panel is demonstrated on the optimization of RNA isolation procedure from biofluids. We also developed a protocol for simultaneous reverse transcription of mRNA and miRNA molecules in a single tube. In combination with direct lysis of cells in plates the protocol represents very time and cost-effective approach for quantification of miRNA and mRNA expression in in-vitro experiments.
New Versatile Enzymes for High Performance Multiplex RNA Detection and Analysis
QIAGEN Beverly, United States of America
Reverse transcription PCR (RT-PCR) and variations like quantitative RT-PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR) are indispensable tools that are widely used for monitoring expression levels of disease-specific mRNA biomarkers, profiling noncoding RNA, and detecting pathogens, especially RNA viruses, at high specificities and sensitivities from biological and environmental samples. Despite their importance, no completely satisfactory RT is currently available. Most one step RT-PCR kits contain a mixture of two enzymes: a retroviral RT, usually a M-MLV derivative, and a thermostable DNA polymerase, usually derived from Taq polymerase. This reliance on two enzymes limits the temperature of cDNA synthesis and necessitates compromises in reaction conditions between those favorable for each component, which can hamper detection of certain RNA targets. A molecular screening and directed evolution program has provided single-enzyme hot-start RT-PCR solutions based on a new thermostable RT that is active at up to 85°C and stable up to 95°C. Higher temperature detection improves specificity and sensitivity by allowing direct detection of RNA targets at near-single-copy sensitivity. This is especially notable when detecting rare or highly structured targets. This new chemistry is compatible with singleplex and multiplex formats using most dye and probe-based detection chemistries. Detection facilitated by this enzyme is equally effective on RNA or DNA targets and can allow simultaneous detection of both without modification of the detection protocol. Because cDNA synthesis occurs rapidly during the initial stage of PCR, the elimination of a distinct RT step and inherently faster kinetics reduces detection times to less than 40 minutes. The inherent stability of the enzyme provides simplified formulation and improved liquid and dry storage. These improvements promise to simplify, accelerate, and improve the reliability and flexibility of detection and analysis of mRNA, noncoding RNA, and viral targets.
Polymerase Strand Displacement Reaction with SD DNA Polymerase – New Alternative to PCR
Polymerase Chain Displacement Reaction (PCDR) was described in 2013 by Harris et al (Biotechniques. 2013, 54(2), 93). It is a new technique combining thermocycling (like PCR) and isothermal (like SDA or LAMP) approaches to DNA amplification. For PCDR performing, two or more pairs of nested primers are used with a thermostable DNA polymerase that has strong strand displacement activity. When extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by strand displacement activity.
In contrast to PCR, new technique allows to synthesize several new DNA copies from the single template DNA molecule in each amplification cycle. Thus, PCDR is much faster and more effective than PCR. The assays based on this reaction are more sensitive. Besides, due to the use of several nested pairs of primers the specificity of the reaction is increased.
Till recently the main disadvantage of PCDR was the lack of commercially available thermostable DNA polymerase which is suitable for PCR and possesses strong strand-displacement activity. SD DNA polymerase (Bioron GmbH Patent US 9,896,671) allows to fill-in this gap and PCDR started to be a reality.
SD polymerase has been shown to direct effectively PCDR in several diagnostics application (Dengue virus detection, plant pathogen HLB detection). Moreover, recently the PCDR on the basis of SD polymerase has been developed further to so-called tailed tandem repeat PCDR (TTR-PCDR) which looks quite universal and can be applied for the fast and convenient detection of pathogens (J. Wang, et al. (2018) Anal. Methods (35), DOI:10.1039/C8AY01625A).
Thus, PCDR with SD polymerase is a promising method which outperforms PCR in speed and efficiency of amplification and can be used in laboratory practice for research and diagnostics purposes.
QPCR, DPCR and NGS for The Detection of EGFR Mutations In Lung Cancer Patients
University of Florence, Italy
Lung cancer is a leading cause of cancer death worldwide. Patients with EGFR oncogene driving mutations benefit from treatment with tyrosine kinase inhibitors but eventually progress developing resistance to therapy. The most common mechanism of resistance is the secondary acquired mutation EGFR p.T790M. As a consequence, it is important to assesses EGFR mutational status at the time of diagnosis and during treatment. Molecular testing guidelines for selection of lung cancer patients for tyrosine kinase inhibitors indicate cell-free DNA (cfDNA) as a surrogate for the determination of EGFR status in patients with limited and/or insufficient tissue.
We performed a pilot study to compare different methods for the detection of EGFR mutations in cfDNA from a small cohort of lung cancer patients. In particular, we evaluated the performances of three approaches based on qPCR, dPCR and targeted NGS.
Results were mostly concordant, but some discrepancies were found for qPCR and dPCR. NGS in some cases allowed a more sensitive detection of variants, especially when a tag sequencing approach was adopted. The major advantage of NGS is represented by the possibility of a simultaneous detection of multiple targets. The pre-analytical phase, in particular cfDNA extraction methods, could also influence the results.
From our preliminary data emerges that the combined use of multiple methods allows a more comprehensive and reliable assessment of the presence of mutations in cfDNA.
SMARTer PicoPLEX Gold: A New Generation of Single Cell NGS Library with High Reproducibility, and Greatly Improved Coverage and Fidelity for Precision Medicine
1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA
Accurate, reproducible detection of mutations and copy number variations (CNV) from small amounts of DNA, including single cells and fixed tissue, is key for genetic analysis of clinical samples to assist in identifying the best treatment regimen and molecular diagnoses of diseases such as cancer. Increasing demand for genetic analysis from limited samples, including single cells, has created an increasingly unmet need for technologies that allow for economical and accurate analysis of those samples. A primary application for single-cell analysis is Preimplantation Genetic Testing (PGT). PGT has grown dramatically in the last ten years, enabled by improvements in the array and sequencing platforms, as well as by the patented SMARTer® PicoPLEX® WGA (PicoPLEX) quasi-random priming technology―the international gold standard for whole genome amplification (WGA) for subsequent detection of CNV in fixed or unfixed single cells. Initially, PicoPLEX chemistry was optimized to allow for reproducible detection of aneuploidies and CNVs in embryo biopsies. The original versions of the technology are not optimized for other applications such as genetic analysis in cancer screening, diagnosis of disease, or therapeutic drug monitoring due to the frequency of falsepositive mutation rates. To address the need for accurate detection of single nucleotide variants (SNVs), we enhanced the PicoPLEX chemistry using optimized enzymes, primers, and protocols that improve sequencing coverage, uniformity, and accuracy while increasing the resolution for CNV detection and retaining reproducibility. This enhanced chemistry named SMARTer PicoPLEX Gold Single Cell DNA-Seq Kit (PicoPLEX Gold) is a single-cell library-prep kit with a simple, four-step protocol to convert fixed or unfixed single cells into NGS libraries in under three hours with minimum hands-on-time. Libraries prepared from single GM12878 cells using the PicoPLEX Gold kit were sequenced on an Illumina® NextSeq® platform to a depth of ~35 million read pairs (2 x 150 cycles), generating >50% genome coverage. This coverage represents a 2-fold improvement over the original PicoPLEX kit, along with a 4X reduction in duplication rates. The PicoPLEX Gold kit detected 3.5X more SNVs compared to Multiple Displacement Amplification (MDA) with the same number of reads. Our proprietary highfidelity polymerases used in the PicoPLEX Gold kit produced up to 50% lower allele dropin (false-positive) rates than MDA. The increased coverage and low bias of the PicoPLEX Gold kit translated to extremely low allele-dropout rates (ADO), ~5X lower than MDA. Therefore, a single PicoPLEX Gold kit library enables reliable, high-resolution CNV analysis with shallow sequencing, and an accurate and reproducible SNV and CNV analysis with deeper sequencing.
Sample quality control of Cell-free DNA
Agilent Technologies, Germany
Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on preanalytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns.
SiPOOL: Fast, Reliable Gene Silencing With Exceptional Target Specificity Using Optimally-Designed Complex siRNA Pools
1siTOOLs Biotech GmbH; 2University of Regensburg
RNA interference (RNAi) is widely used as a gene silencing tool to determine gene function. This is due to its ease of application, broad cell type applicability, drug-like properties and quick time to results. However, it is widely established that synthetic RNAi mediators, short interfering RNAs (siRNAs), can produce wide-spread off-target effects. This gives rise to highly variable results, necessitating time-intensive and costly validation efforts with multiple siRNA reagents. False positive results from siRNA off-targeting may also incur significant costs if left undetected. siPOOLs are complex pools of 30 optimally-designed siRNAs against a single gene. The high complexity pooling reduces the concentration of individual siRNAs, diluting siRNA-specific off-target effects. In contrast, target gene knock-down efficiency is increased due to the greater transcript coverage offered by a siPOOL. As a result, loss-of-function phenotypes become more robust and reproducible. siPOOL rescue constructs were also demonstrated to work efficiently to restore gene function. siPOOLs are ideal tools to ascertain gene function quickly and reliable and can be used in high-throughput RNAi-based screens to identify novel targets across various cell-based systems.
Small Circular RNAs With Antisense Function
Justus Liebig University of Giessen, Germany
Circular RNAs (circRNAs) are a relatively new class of non-coding RNAs comprised of hundreds of members, which are generated from pre-mRNAs by alternative splicing and are present in all eukaryotes investigated so far. Although a defined function of circRNAs is still under debate, some studies revealed a microRNA sponge function of naturally occurring circRNAs like ciRS-7 and SRY. In addition, several other, hypothetical roles have been discussed, such as templates for translation into peptides or proteins, protein sponging, allostery, scaffold functions in RNA-protein complex assembly, or antisense activity.
Here we describe a method for inhibiting translation initiation by small circular antisense RNAs. Translation of mRNAs is one of the mechanisms through which protein levels can be selectively regulated. The eukaryotic initiation factor eIF4E is one of the key regulators, which recognize the cap and forms a trimeric complex (termed elF4F) which is responsible for the activation of mRNA during the translation. This mRNA activation and also the translation can be inhibited by blocking the elF4F complex by binding of small antisense circRNAs to the 5'-untranslated region of mRNA, thereby impeding ribosome attachment and subsequent scanning of the mRNA. As a proof of principle, we designed and generated small synthetic antisense circular RNAs by in vitro transcription and ligation. HeLa cells were co-transfected with synthetic antisense circRNAs targeting ß-globin-luciferase-reporter constructs. Luciferase reporter assays were performed to measure translation activity in transfected HeLa cells. We could reach a translation reduction up to 60 %. Our results with synthetic designer circRNAs establishes that circular RNAs can function as antisense vectors, suggesting that natural examples of antisense circRNAs may exist. Furthermore, this suggests a possible therapeutic use of small circRNAs to inhibit translation.
Streamlined Single-tube Solutions for High Quality DNA Library Preparation
Quantabio, United States of America
Simple, rapid, and reliable solutions for library preparation are in great demand as the next-generation sequencing (NGS) technology has become widely adopted in research, clinical and many other fields. Leveraging our innovative chemistries, world-class enzyme purity and rigorously controlled production and ISO13485 quality system, we have developed several high-quality library preparation solutions for Illumina platforms to address the speed, performance, and throughput requirements of different DNA-seq applications. The sparQ DNA Frag & Library Prep Kit supports easily tunable enzymatic fragmentation and end polishing in a single step, followed by high efficiency adapter ligation in the same tube without intervening purification steps. The simplified workflow combined with the novel chemistry lead to shorter hands-on time (30 minutes), higher library yield (up to 2 folds), better sensitivity, lower duplication rate, and greater coverage uniformity across a broad range of GC-content. For sample types not requiring initial DNA fragmentation, the sparQ DNA Library Prep Kit provides a fast, single-tube library preparation solution that ensures great sensitivity (down to 250 pg) and efficiency, while maximizing library yields. A high efficiency, low bias and high-fidelity PCR master mix was also developed to address applications that require accurate library amplification.
TATAA Biocenter - Your Full Range Commissioned Service Provider. Get Better Results Faster
TATAA Biocenter, Sweden
TATAA Biocenter offers full range of commissioned services and research projects, custom designed for your needs within the area of nucleic acid analysis (qPCR, dPCR, NGS, isolation/extraction, data interpretation) and more. We are a biotechnology company working at the forefront of molecular biology research delivering highest quality data using the most appropriate platforms based on more than two decades of experience within the field, including proprietary technologies such as Two-Tailed RT-qPCR and SiMSen-Seq. At TATAA, we also provide active tailored support throughout the whole lifetime of your project with special focus on quality control,,. The work is performed in compliance with the International Standard ISO/IEC 17025:2005.
TATAA participates in the development of standardized procedures for molecular analyses within the EU initiative SPIDIA (www.spidia.eu) and the IMI initiative CANCER-ID (www.cancer-id.eu), of which the forthcoming CEN and ISO guidelines are based.
Example of services:
- Gene expression profiling (RNA-seq, high throughput qPCR)
- qPCR/dPCR assay design and validation
- Rare event detection (dPCR, SiMSen-Seq)
- miRNA profiling (EdgeSeq, Two-tailed RT-qPCR)
- cfDNA analysis (dPCR, qPCR, NGS)
- Copy number variation (dPCR)
- Custom genotyping services (rhAmp and TaqMan chemistry)
- High throughput Protein profiling (PEA chemistry, Olink Proteomics)
- Single cell analysis (CellCelector, 10X)
Our customers find TATAA Biocenter services cost and time effective. Full projects can be outsourced as well as optimizations of workflows and protocols, in which case we train customer’s staff and transfer the methods and technologies to our client for use in their environment.
Androvic et al. “Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification” Nucleic acids research vol. 45,15 (2017): e144.
Ståhlberg et al. “Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing”Nucleic acids research vol. 44,11 (2016): e105.
Bustin et al. “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments”
Clinical Chemistry 55,4 (2009).
Forootan et al. “Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)” Biomolecular Detection and Quantification 12 (2017) 1-6
Björkman et al. “Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity” Biomolecular Detection and Quantification, Volume 6, January 2016, Pages 4–12
The Spectrum Compact CE System: Fragment Analysis with a Personal Capillary Electrophoresis Device
1Promega Corporation, Madison, WI 53711, USA; 2Hitachi High-Technologies Corporation, 882 Ichige, Hitachinaka-shi, Ibaraki-Ken, 312-8504 Japan
The Spectrum Compact CE System, a collaborative development between Promega Corporation and Hitachi High-Technologies Corporation, allows laboratories of all sizes the freedom to carry out single nucleotide polymorphism, PCR sizing and microsatellite analysis, de novo sequencing, NGS validation, and mutation detection. This four-capillary benchtop capillary electrophoresis instrument runs 1-32 samples and features single base resolution and 6-dye detection along with an integrated touch-screen for instrument operation. Pre-filled reagent cartridges, including a choice of separation polymers, streamline instrument setup and support both fragment analysis and sequencing workflows. This instrument is capable of detecting up to 6-colors with analysis using either GeneMapper® ID-X or GeneMarker® HID Software for Spectrum CE System. It is backwards compatible for all commercially available existing STR systems with performance comparable to current capillary electrophoresis instruments (e.g., sensitivity and mixture samples). Total electrophoresis time for fragment analysis is approximately 40-45 minutes. For the sequencing application, Promega has developed the Prototype ProDye™ Terminator Sequencing System. This is a dideoxy dye-terminator based sequencing chemistry that can generate readable sequences using a variety of templates. In combination with the Spectrum Compact CE System, read lengths of ≥ 600 bases can be obtained with run times of ≤ 32 minutes and ≥ 700 bases with a run time of ≤ 60 minutes.
The Synthetic DNA Fragment Use in the qPCR Diagnostics
1National Institute for NBC Protection (SÚJCHBO, v.v.i.), Czech Republic; 2Faculty of Sciences, University of South Bohemia in České Budějovice, Czech Republic
The aim of this presentation is to introduce the approach using a synthetic control sample in the qPCR-based detection strategies and point out the main advantages connected to its use. The term synthetic control is understood as a commercially synthesized double-stranded DNA of different length tailored to your target sequence or gene of interest.
Although the original intention is to offer ready-to-clone DNA fragments, this strategy can be especially useful considering the samples of biological species difficult to access. It could be the case of rare species or highly pathogenic viruses e.g. Ebola virus, SARS virus etc. The acquisition of such samples could be even impossible, or at least time-consuming concerning the complicated paperwork, and also very expensive when considering distant parts of the world. Additional reasons comprise the biosafety and biosecurity issues which are connected with the particular workplace, as well as the national legal restrictions.
To compensate for the lack of available samples, the synthetic control represents a suitable way to validate the assay without a real sample of the target agent. Thanks to the fact that the control can be commercially synthesized within a few days, it is possible to order a new stock of your target very fast and easy. Moreover, the control is prepared highly concentrated ensuring a sufficient amount of working solution. The dilutions can be used as a concentration standard for further assay validation according to the MIQE guidelines. However, certain limitations exist comprising the length, complexity of the sequence, GC content etc.
To support the presented idea, the examples of particular assays will be mentioned, as well as the workflow involving the design of synthetic control, starting from the thorough in silico study based on available target sequences in the Genbank database, up to the order placement. Thus, it is demonstrated an efficient tool for the set up of a synthetic positive control in the detection of uncommon samples or simply as a useful concentration standard for the validation of qPCR assays.
This work was supported by the Ministry of Interior of the Czech Republic, no. VI20172019063.
ThermaGenix’s Universal PCR Additives for Suppressing DNA and RNA Amplification Errors.
Thermagenix, Inc,, Natick, Masaschusetts, United States of America
The Polymerase Chain Reaction (PCR) is a mainstay of molecular biology and genomics. Mispriming errors during setup and PCR compromise the specificity and sensitivity of DNA and RNA amplification and represent a major obstacle for rapid development of accurate PCR tests. ThermaStop™, ThermaGo™, and ThermaStop-RT™ are novel, easy-to-use universal additives that suppress mis-priming errors before, during, and after PCR for reactions starting with either DNA or RNA. Unlike commonly used antibody and chemical-based hot-start reagents, ThermaStop™, ThermaGo™, and ThermaStop-RT™ are not irreversibly inactivated by heat. Instead, these additives renature upon cooling and then bind to all Type A and Type B DNA polymerases (or reverse transcriptases in the case of ThermaStop-RT™) to enhance their specificity in a temperature-dependent manner; see https://www.thermagenix.com/thermastop-test-data.html for a full list of enzyme tested. ThermaStop™ is a hot-start reagent that outperforms hot-start Taq DNA polymerases. ThermaStop™ makes DNA polymerases more specific in their use of correctly matched primers and suppresses primer dimer formation in no template controls and in amplification reactions with target DNA. ThermaStop™ also reduces the time required to build multiplexed reactions, makes multiplexed reactions more accurate and informative, and acts synergistically with standard antibody-based hot-start for both Type A and Type B DNA polymerases. Complete master mixes containing all reaction components, target DNA, hot-start antibody, and ThermaStop™ remain inactivated for days or months, depending on the temperature at which they are stored prior to being heated the first time. ThermaStop™“cold-stop” activityalso preserves the integrity of amplification products after PCR and enhances the quality of all downstream applications, including NGS, deep sequencing, and Gibson assembly. ThermaGo™ acts synergistically with ThermaStop™ to improve amplification specificity. ThermaStop-RT™ is a first-in-class hot-start reagent that controls the low temperature activity of MMLV and AMV derived reverse transcriptases used for cDNA synthesis in One-Step and Two-Step RT-PCR. ThermaStop-RT™ allows for use of less reverse transcriptase, enhances the accuracy of highly multiplexed RT-PCR assays, and permits quantitatively accurate measurements of very low levels of RNA. ThermaStop™, ThermaGo™, and ThermaStop-RT™ deliver High Precision PCR and RT-PCR by promoting more efficient use of primers, higher levels of intended products, and increased sensitivity and quantitative accuracy for both monoplex and multiplexed PCR and RT-PCR assays. ThermaStop™, ThermaGo™, and ThermaStop-RT™ are commercially available from ThermaGenix’s, Inc., Natick, MA.
Use of Single Control DNA to Internally Evaluate Performance of qPCR and dPCR Instruments
National Institute of Biology, Slovenia
Calibrated and properly maintained equipment is the prerequisite for reliable measurements. Maintenance and operational qualification of real-time quantitative PCR (qPCR) and digital PCR (dPCR) equipment can become very costly, particularly when (1) there are several instruments in a laboratory, (2) when checking needs to be done more than once per year or (3) when instruments are rarely used. However, complete operational qualification is not always necessary, e.g. when instruments are moved or minor repairs are done.
Maintenance and operational qualification procedures and their time intervals for instruments have to be defined by each laboratory according to its needs, manufacturer recommendations, frequency of use and knowledge about the instruments. Here we present an approach that enables regular in‑house checks of instruments by trained laboratory personnel.
To enable a successful implementation of this approach we have prepared a control DNA that enables us to check the performance of different qPCR (LightCycler® 480 System, Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System, ViiA 7™ Real-Time PCR System and 7900HT Fast Real-Time PCR System) and dPCR platforms (Bio-Rad QX100™ and QX200™ Droplet Digital™ PCR Systems, including AUTO DG and Fluidigm Biomark™ HD), after minor repairs, as an intermediate check up between operational quantifications or as a yearly check. Following DNA extraction from the reference material (MON87769, AOCS 0809-B), using CTAB method, two European Union Reference Laboratory validated assays targeting (1) soybean reference gene – lectin, and (2) genetically modified soybean line - MON87769, were used for evaluation of control DNA. After operational qualification of QX100™ Droplet Digital™ PCR System, copy number per 1 µL of DNA solution for both amplicons, using singleplex and duplex reactions has been assessed and criteria for acceptance were determined.
To check the performance of dPCR platforms, control DNA is analysed with both singleplex and duplex assays targeting lectin and MON87769. To check the performance of qPCR platforms, standard curve is prepared from control DNA and analysed using singleplex assays only. To observe possible differences between different parts of the cycler block, reactions are spread across a 384-well PCR plate
The presented approach serves as a complement to the external service, enabling us to maintain instruments performance between external qualifications and reduces the cost of checks between external testing.
MOL-PCR and xMAP Technology: Fast Multiplex Method with High Sensitivity
Veterinary Research Institute Brno, Czech Republic
The world of diagnostics goes still forward and puts demands on laboratories especially in diagnostic of important infective agents. Methods for multiplex detection are mainly designed for relatively fast and cost-effective examination of wide spectrum of different analytes to save money and time and give a complex results. xMAP (x=analyte, MAP= Multi Analyte Profiling) technology provides qualitative analysis of various proteins or nucleic acids simultaneously in a single reaction and enables the direct detection, identification and typing of more than 50 different targets from one biological sample.
This technology is based on multiplex oligonucleotide ligation – polymerase chain reaction (MOL-PCR) with an adaptation to xMAP detection system using different sets of microsphere in a liquid suspension. These are dyed of red and infrared fluorophores. The concentration of internal dyes gives to each set its unique spectral address. During MOL-PCR, a pair of probes (MOLigo1 and MOLigo2) designed specific for target sequence binds to a searched part of analyte and the process of their ligation creates templates for amplification in PCR where the universal primer pair (one has a fluorescent label) is used. The specific part of one MOLigo probe contains the unique TAG sequence – oligonucleotide sequence complementary with an anti-TAG sequence covalently coupled on the microsphere surface. Thanks to this xTAG technology, amplified denaturized products hybridize to corresponding microspheres set. Use of different TAG/anti-TAG sequences enables multiplex and qualitative sample analysis in MAGPIX® instrument (Luminex Corporation) where the medium fluorescence intensity (MFI) is measured.
During routine analysis, xMAP technology thus brings an opportunity to very quick identification of diverse targets (from gene expression monitoring to detection of nucleic acids, proteins, saccharides and another macromolecules) using small amount of examined material.
This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.
Exteme One-Step RT-qPCR: Potential for point-of-care viral detection.
Univeristy of Utah, United States of America
Introduction: Reverse transcription (RT) is often coupled with qPCR for simplified detection of RNA. Despite the ease of One-Step RT-qPCR reactions, some pathogens are still assessed using a Two-Step method. Most Two-Step methods require over an hour for pathogen identification. Even the best commercially available One-Step RT-qPCR kits require a minimum of 20 minutes for detection. Due to the essential nature of fast and accurate detection of viral infections, we have developed a One-Step RT-qPCR assays that can be performed in under 1 minute.
Methods: A prototype instrument was used to temperature cycle 5 µL samples in 0.4-1.0 seconds at annealing, extension, and denaturation temperatures. To transition rapidly between temperatures, reactions required increased polymerase, primer, and Mg++concentrations. Gene specific primers were designed with melting temperatures between 60-75˚C. Detection sensitivity and efficiency of 5 different viral targets were analyzed by real-time PCR. Sample specificity was demonstrated by high-resolution melting.
Results: Viral targets acquired from the American Type Culture Collection (ATCC) were amplified as 50- to 100-bp targets in a minute or less. Without the use of hot start methods, the Extreme RT-qPCR assay produced no-template controls with realtively early amplification. This was attributed to the increased reaction component concentrations resulting in enhanced non-templated additions. However, with the implementation of hot start techniques, non-templated additions were significantly reduced prior to thermocycling as confirmed by real-time PCR and high-resolution melting.
Conclusions: Use of Extreme diagnostic technology has led to the design of a One-Step RT-qPCR assay that can be performed in under a minute with competitive sensitivity to alternative RT-qPCR methods. Extreme RT-qPCR illustrates the ability of point-of-care diagnostic testing for infectious disease when prompt results may be critical.
QC Measurements for Predicting Downstream NGS Success with FFPE and Circulating Cell-Free DNA Plasma Samples
Promega Corporation, 2800 Woods Hollow Rd. Madison, WI
Introduction: Quantity and quality of DNA from formalin fixed, paraffin embedded(FFPE) tumor tissue samples is highly variable, with degradation and crosslinking due to the fixation process leading to issues with amplification and difficulty in NGS analysis. An alternative to FFPE is circulating cell-free DNA (ccfDNA) from plasma or other biological fluids. Compared to gDNA, ccfDNA yields are typically low, with tumor cell present at significantly lower frequencies. Due to the inherent variability of FFPE and ccfDNA, knowing the quantity of DNA is not in itself reliably predictive of downstream NGS success. In this poster, we describe novel methods for predicting sequencing result quality utilizing a multiplexed qPCR assay. Methods: DNA was purified from four matching tumor and normal FFPE tissue types as well as ccfDNA from plasma samples using multiple methods. DNA quantity was measured via single-target qPCR and used for downstream NGS library construction with a 56 gene oncology panel and subsequent data analysis. Discrepancies between quantity of DNA input into library preparation and expected library yield and sequencing coverage uniformity were noted. To investigate if downstream library yield and sequencing quality could be better predicted, a multiplexed qPCR assay was designed that included three different amplicon sizes (75, 150, and 300bp). The quantitative differences between the increasingly larger amplicon sizes were calculated as a ratio to determine the level of degradation of the DNA from FFPE samples. ccfDNA fragments cluster around 170bp; thus, the 150bp target closely estimates ccfDNA concentration. Since gDNA is expected to be much larger in size, the ratio of 150bp to 300bp targets can help predict the ratio of ccfDNA to gDNA. FFPE samples with high 75/300bp ratios are indicative of highly degraded samples, and ccfDNA samples with low 150/300bp ratios are indicative of gDNA contamination. Results: Retroactive testing with the multiplexed qPCR assay showed a strong correlation of degraded DNA to low library yield and low coverage uniformity. Samples with equal concentration and library input performed much better when degradation ratios were low. Conclusions: Data derived from a multi-size target qPCR assay can be very effective in predicting downstream NGS success. Using such a QC method can drive researchers to triage samples and make informed decisions about what downstream library method to use. Concentrating on less complex panels or ddPCR vs. highly multiplexed panels or whole exome sequencing for degraded samples can ensure getting the most useful information out of an individual sample, thus saving time, cost, and loss of information about precious samples.
Combination of Hormone and Transcriptome Analyses in the Study of Cold and Freezing Stress in Grass Lolium perenne
1Institute of Experimental Botany, Czech Academy od Sciences, Czech Republic; 2Faculty of Science, Charles University, Czech Republic; 3Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Czech Academy of Sciences, Czech Republic
Plants in temperate zone have to adapt to the changing temperature during year seasons. Cold and freezing temperatures cause misbalance in metabolism (stress). The plant responses to environmental changes are driven by phytohormones. In our study, we have used frost-sensitive and frost-tolerant clones of ryegrass Lolium perenne in order to describe changes in their metabolism associated with high stress tolerance. Plants were exposed to frost treatment (-7°C for 4 d), either directly or after cold acclimation (3°C for 7 d). Samples of leaves, crowns (meristematic tissue) and roots were analysed by UHPLC–ESI–MS/MS to determine the content of 87 hormone metabolites and by RT-qPCR for transcription analysis. The transcription of selected stress-related and hormone-related genes was correlated with the content of phytohormones.
The resistant clone had lower basal level of cytokinins (CKs) as well as the lower transcription of CK biosynthetic genes (LpIPT5 and LpIPT8) in roots. Contrarily, the transcription of stress-related genes LpIRI3 and LpIRI4, and abscisic acid (ABA) biosynthetic gene LpNCED1 was higher than in the sensitive clone. Cold acclimation was associated with elevation of CK levels and transcription of LpIPT genes, especially in crowns. Also, ABA and salicylic acid (SA) levels were up-regulated in all organs. The strong effect of the clone frost resistance was observed in the case of auxin. Subsequent frost led to increase of CKs in leaves, but decrease in crowns. The content of ABA and SA decreased, substituted in roots and crowns by the higher content of jasmonic acid (JA). Direct frost had a negative impact on highly-active CKs and it stimulated accumulation of low-active forms, as well as ABA and SA. The level of JA was in this case suppressed in roots and crowns but highly up-regulated in leaves. The transcription pattern of hormone-related genes generally correlated with the levels of phytohormones, few exceptions will be described. The transcription of stress-related genes differed significantly between tolerant and resistant clones, mainly in the case of LpCBF3, LpIRI3 and LpIRI4. In conclusion, our results show that the content of phytohormones differs dramatically in the situation of direct frost or freezing after acclimation. The regulation of the responses is in large part caused by different regulation of hormone metabolism and signalling.
Acknowledgement: The work was supported by the Czech Science Foundation, project no. 17-06613S.
Differentiation Potential of NG2-glia Following Different Types of Brain Injuries
1Institute of Biotechnology, Czech Republic; 2Institute of Experimental Medicine, Czech Republic; 3Faculty of Science, Palacký University, Czech Republic; 4Faculty of Science, Charles University, Czech Republic; 5TATAA Biocenter, Sweden
NG2-glia, a fourth major glial cell population, are present in the adult central nervous system and display distinct morphology, antigens and functions from other mature glial cell types. Recently, many studies have shown that these cells are multipotent in vitro and they also display wide differentiation potential under pathological conditions in vivo. The aim of this study was to identify differentiation capacity and rate of proliferation of NG2-glia after different types of brain disorders, such as focal cerebral ischemia (FCI), stab wound (SW) and demyelination (DEMY). We used transgenic Cspg4-cre/CAG-tdTomato mice, which after administration of tamoxifen express red fluorescent protein (tdTomato) in NG2-glia and cells derived therefrom. Differentiation and proliferation potential of tdTomato positive (tdTomato+) cells in sham-operated mice and those after injury were determined using single cell RT-qPCR and immunohistochemistry. We identified two main populations (NG2-glia, oligodendrocytes) which each had 4 subpopulations. Expression profiles showed that one subpopulation (astrocytic like NG2-glia) were present only after FCI not after DEMY or SW what confirmed our data of immunohistochemistry. Moreover, this subpopulation had a high percentage of cells expressing Smoothened, a key factor of Shh signaling. Taken together, we have described the ability of NG2 cells to acquire multipotent phenotype after different types of injury and that the phenotype fluctuates depending on the pathological condition.
Metrological Support for Molecular Diagnostics - A Case Study of HCMV and Its Resistance to Ganciclovir
1National Institute of Biology, Department of Biotechnology and Systems Biology, Slovenia; 2Biotechnical Faculty, Department of Biology, University of Ljubljana, Slovenia
Methods for fast, accurate and sensitive detection, identification and quantification of pathogens are of paramount importance for reliable and effective disease control. While nucleic-acid amplification‑based methods offer great potential in clinical diagnostics, reproducibility and comparability of results are hampered due to the lack of reference methods and reference materials. One of the challenges of metrology, the science of measurement, is the establishment of support for reliable measurement of nucleic acid concentration.
To investigate the potential of application of metrology in the development of methods for molecular diagnostics, resistance of human cytomegalovirus (HCMV) to antiviral drug ganciclovir (GCV) was selected as a case study. HCMV or human herpesvirus 5 (HHV5) is a 230 kB double stranded DNA virus infecting monocytes, macrophages and dendritic cells. HCMV often leads to wide spread of viral replication and dissemination to multiple organs and can be life threatening to individuals with compromised or immature immune system. Antiviral drugs, predominantly GCV, are used to treat patients and prevent further dissemination of the virus. As the development of resistance can be life threatening especially for immunocompromised patients, a reliable method for detection and monitoring of infection and viral load is necessary.
The aim of our research was to develop and assess in terms of sensitivity, repeatability, linearity, dynamic range and robustness, nucleic-acid amplification‑based methods targeting three of the most common point mutations that contribute to GCV resistance in HCMV. In recent years digital PCR (dPCR), has become widely used in the field of nucleic acid analysis, including viral diagnostics. Although, dPCR is a prosperous methodology, the majority of diagnostic laboratories rely on real-time quantitative PCR (qPCR). Thus, the developed methods were assessed both on dPCR and qPCR platforms in order to determine the suitability of different platforms for molecular diagnostics. This research was financially supported by the AntiMicroResist project (the EMRP project, which is jointly funded by the EMRP participating countries within EURAMET and the European Union).
Detection of Bacteria and Virus Involved Urinary Tract Infections and Profiling Their Antibiotic Resistance Using TaqMan Real-time PCR Assays
Thermo Fisher Scientific, United States of America
Urinary tract infections (UTI) are commonly caused by the gram negative bacteria Escherichia coli, Klebsiella pneumoniae and the gram positive Staphylococcus epidermidis, and Enterococcus faecalis. Other less frequent bacteria causing UTIs are Morganella morganii, Acinetobacter baumanii, Citrobacter freundii and others. Some viral and fungal pathogens also have been associated with UTI.
We developed comprehensive panel of real time PCR assays to detect different bacteria, virus and fungi involved in UTI. The panel is based on the TaqMan® assay platform and was designed to allow the specific detection of these pathogens on the microfluidic OpenArray® platform. A bioinformatic assay design pipeline was developed to enable the design of specific and sensitive detection assays taking under consideration genome sequences of 80,000 bacterial species deposited at NCBI.
UTI infections are often resistant to antibiotic treatment. Carbapenem resistant and extended spectrum beta lactamase harboring bacteria are most commonly found in UTI. Using our assay design pipeline we also developed a panel of TaqMan® assays to detect genes conferring antibiotic resistance. We focused on beta lactamases and included detection assays for a wide variety of different members of this group, including the OXA, PER, NDM, CTX and other beta lactamases. Also assays for predictive identification of vancomycin resistance and quinolone resistance are included. The combined panel allows the identification of UTI associated microorganisms and their antibiotic resistance genes on the microfluidic OpenArray®system.
Immunomodulatory Properties of Adipose-Derived Stem Cells Treated with 5-Azacytydine and Resveratrol on Peripheral Blood Mononuclear Cells and Macrophages in Metabolic Syndrome Animals
1Wrocław University of Environmental and Life Sciences, Poland; 2Faculty of Veterinary Medicine, Equine Clinic-Equine Surgery, Justus-Liebig-University, 35392 Gießen, Germany
Endocrine disorders, including equine metabolic syndrome (EMS), are a serious issue in veterinary medicine and horse breeding. Furthermore, EMS was shown to affect the cytophysiological properties of adipose-derived stem cells, reducing their therapeutic potential. However, it was shown that those cells can be rejuvenated while using a combination of two chemicals: 5-azacytydine (AZA) and resveratrol (RES).In the present study, we decided to evaluate the immunomodulatory properties of AZA/RES-treated adipose-derived stem cells (ASC) isolated from EMS horses (ASCEMS). Thus, we co-cultured ASC with peripheral blood mononuclear cells (PBMC) and RAW264.7 macrophages. Most attention was placed on regulatory T lymphocytes (TREG), as well as the messenger RNA (mRNA) and protein levels of several cytokines(tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-10, and IL-1β). Moreover, we also investigated the expression of genes related to auto- and mitophagy in both PBMCs and ASCs. PBMCs were obtained from healthy and EMS-suffering individuals and were co-cultured with ASCs that were isolated from healthy and EMS horses cultured in control conditions and with AZA/RES. We discovered that cells treated with AZA/RES increase the TREG number while co-cultured with PBMCs. Moreover, the co-culture of PBMCs with AZA/RES-treated ASCEMS induced mitophagy in PBMCs. Furthermore, ASCEMS pre-treated with AZA/RES displayed anti-inflammatory properties, as decreased levels of TNF-α, nitric oxide (NO), and IL-6 were observed in those cells in comparison with their untreated counterparts in the co-culture with RAW264.7 macrophages. In summary, we demonstrated that ASCEMS treated with AZA/RES displayed increased anti-inflammatory properties, and was able to regulate and activate the TREG-related anti-inflammatory response.
Elucidation of the Role of Tenacibaculum spp. in Atypical Winter-ulcer in Sea-farmed Atlantic Salmon in Norway
Norwegian Veterinary Institute, Norway
The disease ‘tenacibaculosis’ otherwise known as ‘atypical winter ulcer’ has become increasingly common in Norwegian Atlantic salmon farming within the last decade. This disease is primarily characterised by severe tissue -necrosis and -loss affecting the head and/or fins of infected fish. Losses related to tenacibaculosis are typically acute and may be extremely high. Incidences of up to 80% mortality are not unheard of and in severe cases affected stocks may be destroyed due to welfare concerns and to avoid possible transmission of infection following the precautionary principle.
The reasons for the recent emergence of tenacibaculosis as a common and severe disease in Norwegian salmon farming remain unknown. It is, however, well recognised that most diseases affecting farmed fish are a result of the interplay between environment, host species and pathogenic agent i.e. the presence of the ‘pathogen’ alone may not necessarily lead to manifestation of disease.
Tenacibaculum species play an important ecological role in recycling marine detritus and more than 25 species have been validly described, with many more remaining as yet unstudied. In an effort to identify a possible aetiological role of particular Tenacibaculum species/strains in development of ‘atypical winter-ulcer’ we sequenced 90 Tenacibaculum isolates collected from 15 geographically distinct disease outbreaks on a MiSeq. The primary aim was to generate a phylogeny over the strains including publically available Tenacibaculum sequences in NCBI.
Investigation Of Association Of Variant Rs17036314 Of PPARG Gene And Rs7923837 Of HHEX Gene In Diabetes Mellitus Type 2 Patients In Iran Population By Tetra-ARMS PCR And Sanger DNA Sequencing
IAUPS, Iran, Islamic Republic of
In recent decades, Diabetes mellitus type 2 is a complex metabolic disease developed by interactions between various genetic factors and environmental factors in adult individuals of Iranian population of variable ethnicity. In this study, genetic investigation of PPARG and HHEX genes in association with diabetes mellitus type 2 disease is elaborated. Genetic study of the association of rs17036314 in PPARG and rs7923837 in HHEX genes with diabetes mellitus type 2 disease is performed by Tetra primer ARMS PCR technique, a frequently used, precise, low cost method for detection of mutations in single nucleotide polymorphisms in involved genes. Study of rs17036314 in PPARG gene (G>C) in 70 patients affected by diabetes mellitus type 2 and 70 normal subjects indicates 54.28% of patients with mutant allele and 45.71% with normal allele. Study of rs7923837 in HHEX gene (A>G) in 70 patients affected by diabetes mellitus type 2 and 70 normal subjects indicates 58.35% of patients with mutant allele and 13.6% with normal allele. Tetra primer ARMS PCR for genotype determination of rs17036314 in PPARG and rs7923837 in HHEX genes is benefited for selection of proper drug therapy, better prognosis of disease, risk assessment of diabetes mellitus type 2 and diagnosis of the disease.
Microvesicles Derived From Human Adipose-Derived Multipotent Stromal Cells Improves Retinal Functionality in Dogs With Retinal Degeneration.
13Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences Wroclaw 50-375, Poland; 2Przychodnia weterynaryjna Trzebnicka; 3Przychodnia weterynaryjna Viva; 4Professor Emeritus of Veterinary Ophthalmology, Royal Veterinary College, London, UK; and Ophthalmology Referrals, 25, Great North Road, Brookmans Park, Herts. AL9 6LB, UK; 5Faculty of Veterinary Medicine, Equine Clinic-Equine Surgery, Justus-Liebig-University, 35392 Gießen, Germany
The aim of the study was to determine the influence of microvesicles (MVs) derived from multipotent stromal cells isolated from human adipose tissue (hASCs) on retinal functionality in dogs with various types of retinal degeneration. The biological properties of hASC-MVs were determined using an in vitro model of retinal Muller-like cells (CaMLCs). The influence of hASC-MVs on cell viability and metabolism and brain-derived neurotrophic factor (BDNF) was tested. Biological activity of hASC-MVs was performed under normal and oxidative stress conditions. Preliminary clinical studies were performed on nine dogs with different types of retinal degeneration. The clinical studies included behavioural tests, fundoscopy and electroretinography before and after hASC-MVs intravitreal injection. The in vitro study showed that CaMLCs treated with hASC-MVs were characterised by improved viability and mitochondrial potential, both under normal and oxidative stress conditions. Additionally, hASC-MVs under oxidative stress conditions reduced the number of senescence-associated markers, correlating with the increased expression of BDNF. The preliminary clinical study showed that the intravitreal administration of hASC-MVs significantly improved the dogs’ general behaviour and tracking ability. Furthermore, fundoscopy demonstrated that the retinal blood vessels became less attenuated and electroretinography using HMsERG demonstrated an increase in a- and b-wave amplitude after treatment. These results shed promising light on the application of cell-free therapies in veterinary medicine for retinal degenerative disorders treatment.
Strigolactone Mediated Age - And Organ-Specific Gene Expression In Arabidopsis thaliana
1Institute of Experimental Botany, Czech Acad. Sci.,; 2Charles University in Prague, Faculty of Science, Department of Experimental Biology of Plants
Inorganic phosphate (Pi), one of the most important macronutrients for plants, is often difficult to obtain due to its low solubility and a general lack in the soil. Nutrient stress conditions represent a serious threat to the environment and agriculture. Strigolactones (SL) are phytohormones, which orchestrate plant responses to the lack of Pi, including regulation of shoot branching and root architecture. The effect of synthetic SL analogue GR24 on gene expression was investigated in Arabidopsis thaliana during Pi starvation and in full Pi nutrition (100 µM). Plants were grown in hydroponics at the presence of 5 µM GR24 in a 7-day experiment. Genes of interest included 3 groups: SL-related; Pi transporters and the antioxidant system-related ones. The aim has been to compare transcriptomic responses in younger (3-week) and older (5-week) plants. The focus was also given to organ-specific (leaves and roots) expression patterns.
GR24 application at full Pi nutrition led in leaves to over-all up-regulation of strigolactone-related genes in older plants, when compared to the younger ones. In roots, more profound up-regulation of several biosynthetic genes (D27; MAX1) and transport ones (PDR1/PDR2) was observed after GR24 application also in older plants. Repressors of SL signal transduction SMXL2/6/8 were up-regulated as well, which might have been caused by positive-feedback regulation of strigolactone signalling pathway. The data suggest that the responses of older plants to exogenous strigolactone GR24 are stronger than those of younger ones. Antioxidant system-related genes were also more up-regulated after GR24 application in older plants, both in leaves and in roots. Phosphate transporters showed a different response in leaves, where PHO1 was down-regulated in older plants, while the opposite trend was found in roots. Also, both PHT transporters (PHT1.4; PHT1.7) were up-regulated in roots after GR24 application. These GR24 responses were strengthened at Pi deficiency.
Results suggested that older plants are not only more sensitive to exogenous GR24 application but that these transcriptomic changes are associated with up-regulation of antioxidant system-related genes. Moreover, we observed that transcriptomic response to GR24 treatment was organ-specific.
Acknowledgement: The study was supported by the Charles University, project GA UK No 1086217.
Role of Nitric Oxide During Embryonic Wound Healing
Laboratory of Gene Expression, Institute of Biotechnology, Czech Academy of Science, Czech Republic
The long-term goal of our team is to determine the mechanism of embryonic wound healing and regeneration. In adults, wound healing is closely associated to the role of nitric oxide (NO), which is a known regulator of inflammation, angiogenesis and matrix metalloproteinases. However, at a certain stage of development embryos lack an immune system and vascular system but are still able to have complete healing of wounds. Herein we present the role of NO in embryonic wound healing.
We used early embryos of Xenopus leavis as our model to study wound healing. Utilizing RNA-Seq methodology, we determined changes of gene expression during this process and using functional assays the effects after inhibition of NO production.
Inhibition of the NO pathway during and after injury of X. laevis embryos led to defective wound healing. Only a small number of genes were found to be involved in the response to injury and were altered when NO was inhibited. Some of these genes (i.e.: aqp3, mmp8, lep) are known to be connected with defence response, which is very interesting given that the embryo is lacking an immune system.
We concluded that NO is important for the regulation of embryonic wound healing and the regulation of embryonic pseudo-inflammatory system.
Study Of An In Vitro System For Alzheimer’s Disease
1Bio-Rad Laboratories, Hercules, CA, USA; 2Bio-Rad Laboratories, Mississauga, ON, Canada
Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, which accounts for over 80% of dementia cases in people over 65 years old. The rapid increase in AD cases not only has a significant impact on the economy, but also imposes huge burdens on families and society due to the disease rendering patients dependent upon others for their health and safety. Many disease models have been established to study AD, including animal models, primary cultures, stem cells models, and cancer cell models (Carolindah et al. 2014). We sought to establish an AD model system in our lab, using human neuroblastoma cells (SH-SY5Y) which can be differentiated into mature neuron-like cells. In order to lidate this system, we used microscopy, RT-qPCR, and multiplex immunoassays to study the expression of key neural biomarkers, including AD markers. Additionally, we screened a long noncoding RNA panel to identify lncRNAs that are differentially expressed during the transition of these cells from neuroblastoma to neuronal cells. However, during the course of our study, we uncovered significant differences between the two SH-SY5Y cell lines we were working with. The two cell lines were sourced from two different suppliers, and we found they were actually very different to each other in terms of gene and protein expression and cytokine secretion.
The Expression of Prostaglandins during different Physiological stages in the Bovine Corpus Luteum
1University of Prishtina, Kosovo; 2Animal Physiology & Immunology Weihenstephan, Technical University of Munich, Germany; 3Department of Veterinary Sciences, Ludwig Maximilian University of Munich, Germany
The ovarian cycle in ruminants is characterized by repeated patterns of specific cellular proliferation, differentiation and transformation that accompanies follicular development and corpus luteum (CL) formation and function. The objective of this study was to characterize some prostaglandin family members in bovine CL during estrous cycle and pregnancy. The CL tissue was assigned to the following stages of estrous cycle: 1-2, 3-4, 5-7, 8-12, 13-16, >18 days (after regression) and of pregnancy: 1-2, 3-4, 6-7 and >8 months. In these samples we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE) and their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS) and PTGE synthase (PTGES). The expression of mRNA was measured by RT-qPCR, hormones by EIA and localization by immunohistochemistry. The mRNA expression of COX-2 shows a clear regulation, with the highest level during early luteal phase (days 1-2) and than a significant decrease during mid luteal phase (P<0.05), with a slight increase during luteal regression (days >18). During pregnancy, the expression level was low agai. The mRNA expression PTGFS and PTGES in CL during early lutel phase (day 1-7) was high (P<0.05), followed by a continuous and significant down-regulation (P<0.05) to a lower plateau in CL during mid, late and regression as well as during all phases of pregnancy. The mRNA expression of PTGFR was high during all phases of estrous and pregnancy with prompt and significant downregulation (P<0.05) just during CL regression. In contrast, the mRNA of PTGER4 was low during all examined phases of estrous and pregnancy with clear significant upregulation (P<0.05) just during CL regression. The PTGER2 mRNA expression data showing no obvious differences in expression during different phases of estrous cycle and pregnancy. Immunohistochemically, the large granulose–luteal cells show strong staining for COX-2 and PTGES during early luteal stage, followed by lower activity afterwards. During pregnancy, most of the luteal cells were only weak positive or negative. In conclusion, our results lead to the assumption that the examined prostaglandin family members (COX-2, PTGFS, PTGES, PTGF, PTGE, and their receptors PTGFR, PTGER2, PTGER4) are involved in the local mechanisms regulating luteal function, namely during CL formation, function and regression and during pregnancy in the cow.
Use of Universal ProbeLibrary Probes for Detection of Highly Pathogenic Biological Agents
National Institute for NBC Protection (SÚJCHBO, v.v.i.), Czech Republic
In the world of the real-time PCR, there are two approaches how to obtain a fluorescent signal from the tube. Some investigators insist that unspecific intercalating dyes are a sufficient and cost-effective system to obtain a reliable signal. Others feel that only probe-based assays are the right approach. The UPL probes (Universal ProbeLibrary; Roche) offer a compromise that provides the specificity of a hydrolysis probe but does not require a unique probe for each target. The UPL probes use a nucleotide chemistry called LNA (Locked Nucleic Acid), which allows very short (8-9 bases) oligonucleotides to be effective in hybridization with the target. LNA allows the melting temperature of the short probes to be unusually high.
Hence, the objective is to demonstrate the use of the UPL probe for the detection of highly pathogenic biological agents. Moreover, we routinely combine the UPL probes with an intercalating dye (SYTO 61 dye; Invitrogen) for the verification of results. The detection strategy based on the use of the UPL probes was validated on various bacteria e.g. Brucella sp., Vibrio cholerae, Salmonella Typhi, Chlamydophila psittaci, further on selected Rickettsiae, as well as in the detection of viruses using RT-qPCR e.g. Chapare, Chikungunya, Ebola, Hantaan, Lujo, Seoul or Sin Nombre virus. Thanks to the shortness of the UPL probes sequences, it is possible to combine one UPL probe with multiple loci (in the combination with different pairs of primers). This feature was used for the detection of both Zaire and Sudan ebolavirus and for the detection of both the Brucella genus and Vibrio cholerae by the probes no. 138 and no. 11, respectively. All the presented assays were validated according to the MIQE guidelines.
In summary, the UPL probes can be used for the rapid, flexible and reliable detection and quantification of highly pathogenic biological agents. Furthermore, it is able to easily design gene-specific quantification assays using the UPL probes and the free web-based ProbeFinder software at the Assay Design
This work was supported by the Ministry of Interior of the Czech Republic no. VF20112015013 and VI20172019063
Using In Vitro Transcribed MRNA For Gene Therapy Targeting Viral Infections
Institut für Virologie, Technische Universität München, Germany
Current gene therapy approaches mainly consist of lenti-, adeno- and adeno-associated viral vectors to introduce genes of interest into cells to reconstitute functionality of missing or defective factors. This approach has clear advantages, such as long-term gene expression after single administration or tissue specific delivery enabled by the tropism of the viral capsid, however there are safety concerns which complicate clinical translation. These include the risk of carcinogenesis arising from mutational integration, immune responses resulting from viral components or negative effects by long-term gene expression. Preexisting immunity against the used viral vector can render this approach ineffective. An approach to overcome these caveats can be the transfection of in vitro transcribed (ivt) messenger RNA (mRNA) that is transiently expressed in targeted cells or tissue. In contrast to viral vectors, this approach lacks viral components and the risk of unspecific integration into the genome.
One potential application is an mRNA based CRISPR/Cas9 system to address viral infections like chronic Hepatitis B by targeting the episomal viral genome (covalently closed circular DNA) or host factors involved in viral lifecycle. We produced ARCA capped Streptococcus pyogenes (Sp) Cas9 protein and ivt sgRNAs which target multiple sequences and genomic loci. In differentiated HepG2-NTCP, HepaRG and primary human hepatocytes mRNA transfection efficiencies were up to > 80 % with high cell viability (>95 %). Introduction of modified nucleotides (e.g. Pseudo-UTP, 5mCTP) significantly increased cell viability, protein expression and functionality. Targeting host genes (e.g. GAPDH or DNMT1) using SpCas9 mRNA showed genome editing that resulted in reduction of protein expression. Preliminary data in HepG2.2.15, a cell line harboring an integrated HBV genome, showed a significant reduction in HBe antigen expression 5 days after transfection of SpCas9 mRNA using sgRNAs against multiple loci in the HBV genome. Significant reduction of viral DNA expression after treatment with SpCas9 mRNA was detected via qPCR. In conclusion, our data indicate that mRNA-based gene therapy is a versatile tool for many in vitro approaches allowing fast and transient expression of SpCas9 or other proteins in dividing and non-dividing cells. Furthermore, ivt mRNA is a promising application for safe anti-viral therapy (as shown for HBV) that can be optimized for in vivo studies and clinical translation.
Whole Genome Crispr Screening to Identify Potential SERD Molecule Resistance Mechanisms
Metastatic breast cancer results in substantial morbidity and mortality for women afflicted with this disease. Approximately 80% of breast cancers express the estrogen receptor. The cancer cell survival and proliferation is driven by the activation of the estrogen receptor. SAR439859 is an effective endocrine therapy for breast cancer selectively and effectively degrading the estrogen receptor. A phase 1/2 study of SAR439859 alone and/or in combination with palbociclib in postmenopausal women with estrogen receptor positive advanced breast cancer was initiated last year by Sanofi.
CRISPR/Cas9 is a recent and revolutionary technology for efficient and directed alterations of the genome. CRISPR allows users to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs (SgRNA). These double strand breaks can be repaired by non-homologous end joining DNA repair mechanism. This DNA repair mechanism is an error prone DNA repair process which can lead to gene invalidation.
We aimed at investigating the potential SAR439859 resistance mechanisms. A better understanding of resistance mechanisms is needed to overcome this potential problem and to propose complementary therapeutic solutions. We conducted a whole genome CRISPR screening in a breast tumor cell line with a thermofisher commercial SgRNA library (ref: M04305) to identified genes involved in potential SAR439859 resistance phenomena. CRISPR screening steps will be presented from the cell line selections to results analysis.