Multiplex Preamplification Research Articles

Investigation of the impact of biogeographic ancestry on DNA methylation based age predictions comparing a Middle East and a Central European population

DNA methylation based age prediction is a new method in the toolbox of forensic genetics. Typically, the method is applied in the course of police investigation e.g. to predict the age of an unknown person that has left a biological trace at a crime scene. The method can also be used to answer other forensic questions, for example to estimate the age of unknown human bodies in the course of the identification process. In the present study, we tested for a potential impact of biogeographic ancestry (BGA) on age predictions using five age dependent methylated CpG sites within the genetic regions of ELOVL2, MIR29B2CHG, FHL2, KLF14 and TRIM59. We collected 102 blood samples each from donors living in Iraq, Middle East (ME) and Germany, Central Europe (EU). Both sample sets were matched in sex and age ranging from 18 to 68 years with exactly one male and female sample per year of age. All samples were analyzed by bisulfite pyrosequencing applying a multiplex pre-amplification strategy based on a single input of 35 ng converted DNA in the PCR. For the CpGs in MIR29B2CHG, FHL2 and KLF14, we observed significantly different methylation levels between the two populations. While we were able to train two highly accurate prediction models for the respective population with mean absolute deviations between predicted and actual ages (MAD) of 3.34 years for the ME model, and 2.72 years for the EU model, we found an absolute prediction difference between the two population specific models of more than 4 years. A combined model for both populations compensated the methylation difference between the two populations, providing MADs of prediction of only 3.81 years for ME and 3.31 years for EU samples. In total, the results of the present study strongly support the benefit of BGA information for more reliable methylation based age predictions.

A Multiplex Thyroid-Specific Assay for Quantification of Circulating Thyroid Cell-Free RNA in Plasma of Thyroid Cancer Patients.

BackgroundThe standard of care for thyroid cancer management is thyroidectomy and adjuvant radioactive iodine (RAI). There is a paucity of clinical tool that quantifies residual thyroid volume reliably for precise adjuvant RAI dosing. Serum thyroglobulin (TG), tumour marker for thyroid cancer, takes 4 weeks for complete clearance due to its long half-life, and might be undetectable in 12% of structural disease patients. It detects recurrence with a sensitivity of 19–40%, mainly attributed to issue of TG antibody interference with TG immunometric assay. We hypothesise that the quantity of thyroid-specific cell-free RNA (cfRNA) is indicative of amount of thyroid tissues, and that during thyroid surgery, cfRNA levels decrease accordingly.MethodsWe identified 11 biologically significant and highly expressed thyroid-specific targets from Human Protein Atlas and literature. To assess for a fall in thyroid-specific cfRNA level, we recruited 16 patients undergoing thyroid surgery or RAI for malignant or benign thyroid disease, and tracked longitudinal trend of cfRNA. To assess the utility of cfRNA in detecting metastatic thyroid cancer, cfRNA of 11 patients at intermediate to high risk of recurrence was measured during surveillance and at time of clinical recurrence.ResultsThe multiplex assay was capable of amplifying and quantifying multiple thyroid-specific genes in a single reaction. The selected targets were amplified successfully from RNA extracted directly from the thyroid (positive control), indicating that they were highly expressed within thyroid tissue. These cfRNAs were present in plasma, in amounts quantifiable using qRT-PCR. Four cfRNA transcripts (TPO, GFRA2, IVD, TG) fell post-treatment in more than 50% of cohort. The thyroid peroxidase (TPO) cfRNA fell post-therapy in 63% of cohort by 80%, as early as 1 day post-treatment, supporting the potential role as early indicator of remnant thyroid tissue volume. We demonstrated the clinical relevance of circulating TPO cfRNA by tracking temporal changes in setting of peri-treatment, recurrence, and TG Ab positive state.ConclusionUsing a multiplex pre-amplification approach, the TPO cfRNA was a potential biomarker that can track residual thyroid mass. It can be further optimised for quantification of thyroid volume to guide RAI doses and for detection of thyroid cancer recurrence.

Abstract 832: Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables

Abstract DNA methylation specific PCR is a commonly used method to determine DNA methylation status. However, a well-known risk for PCR, and particularly methylation specific PCR, is carryover contamination from amplification products that can result in false positive PCR results in subsequent tests. This risk can be mitigated using careful technique, facility design, and biochemical methods such as uracil incorporation and subsequent uracil N-glycosylase (UNG) negative selection. Unfortunately, careful technique is highly user dependent, facility design can be cumbersome and expensive, and uracil incorporation is effective for only one step of two-step (nested) PCR reactions and cannot be used with the DNA bisulfite treatment methods used for methylation specific PCR. Our company has developed a system that mitigates PCR product contamination by continuously containing PCR products in plastic consumables and can interface with existing laboratory robotics, real time thermocycling instruments, and cassette electrophoresis gels. Here we utilize this system to compare the sensitivity and specificity of two-step multiplex nested PCR vs. single-step PCR for DNA methylation status detection applications using both qPCR and gel electrophoresis analysis. Published methylation status specific PCR primers that target CpG nucleotides in promotor regions were used to PCR amplify commercially available fully methylated and fully unmethylated DNA. The results demonstrate that a multiplex preamplification using amplicons for the CHAD, GFI1, MX2, NEU1, VWCE genes followed by monoplex nested methylation specific PCRs resulted in a Limit Of Detection (LOD) of 1 to 10 DNA template copies whereas performing just the monoplex methylation specific PCR (no preamplification) resulted in a LOD of 10 to 300 DNA template copies. If DNA isolated from macro dissected FFPE tissue was utilized, positive signals were identified using multiplex nested PCR whereas no signals were identified using single-step PCR. When mixtures of methylated and unmethylated DNA were utilized along with the GSTP gene amplicons, two-step nested PCR using were able to distinguish as low as 1% unmethylated DNA or 2% methylated DNA in a background of the other form, whereas single-step PCR was able to distinguish just 10% unmethylated or 33% methylated DNA under the same conditions. Furthermore, no PCR product contamination was observed upon amplification of samples collected from the external portion of the device demonstrating the PCR products were well contained. Citation Format: Wanyuan Ao, Derek Bosh, Dale Emery, Robert Parry, Nils Adey. Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 832.

Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.

An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor-derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR. This workflow was then applied in metastatic breast cancer (MBC) patients receiving systemic therapies for MBC. In accordance with previous studies, estrogen receptor mutations were more frequently detected in endocrine-treated MBC patients at progressive disease [34.1% (15/44)] than before the start of endocrine therapy [3.9% (2/51); P=0.001]. For a subset of samples, results were compared with analysis of these mutations by Oncomine-targeted next-generation sequencing, which, although requiring a higher cfDNA input, yielded concordant results. The data establish development and validation of a digital PCR workflow for the simultaneous detection of several tumor-derived mutations in minute amounts of cfDNA and show the potential of this workflow for use on archived volume-limited blood samples.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Genome sequencing and conservation genomics in the Scandinavian wolverine population.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

PO-385 DNA methylation profile of castration-resistant prostate cancer cells

IntroductionDuring colorectal cancer (CRC) development several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumour cells that can be utilised for the diagnosis of CRC. Cell-free DNA (cfDNA) in the circulatory system can originate from tumour tissue; therefore the evaluation of tumor-related methylated DNA in plasma can be a simple and promising minimally invasive method for early cancer screening, especially using automated isolation techniques. Our aim was to analyse the methylation level alterations of 4 selected genes along the healthy-adenoma-CRC sequence in plasma samples. Moreover, we aimed to test different preservative blood collection tubes and automated cfDNA isolation method to determine whether these factors influence the DNA methylation patterns.Material and methods121 blood samples were collected in K3EDTA tubes, and manual cfDNA isolation was performed. The methylation pattern of SFRP1, SFRP2, SDC2 and PRIMA1 was determined using MethyLight PCR containing multiplex preamplification step. Further 30 blood samples were collected in Roche Cell-Free DNA Collection Tubes, which was developed for stabilisation of cfDNA with preservative reagents. In case of these samples, in addition to manual isolation, MagNA Pure 96 Instrument was used for automated cfDNA extraction.Results and discussionsElevated methylation of the genes was observed in 70% to 90% of adenoma and CRC patients. Average DNA methylation level was <1% for all four markers in healthy controls, while in CRC samples aberrant hypermethylation of SFRP1, SFRP2, SDC2 and PRIMA1 was observed with methylation percentage values of 21.77%±33.32%; 6.82%±17.1%; 12.06%±24.37% and 13.66%±25.14%. Using combined analysis, the 4 markers were able to distinguish CRC patients from normal with 91.5% sensitivity and 97.3% specificity and could differentiate AD samples from controls with 89.2% sensitivity and 86.5% specificity. Interestingly, Roche blood collection tube and automated cfDNA isolation method influence the detected methylation levels; however, methylation differences still have been found between the clinical groups, especially in case of PRIMA1 and SFRP1 genes.ConclusionOur findings suggest that the 4 markers can be promising epigenetic biomarkers for colorectal adenoma and CRC detection with high sensitivity. Automated isolation procedures may contribute to wide-spread, screening applications of methylation markers; however, further developments are needed.

PO-384 Manual and automated detection of DNA methylation alterations in colorectal neoplasia in circulating cell-free DNA fraction

Introduction During colorectal cancer (CRC) development several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumour cells that can be utilised for the diagnosis of CRC. Cell-free DNA (cfDNA) in the circulatory system can originate from tumour tissue; therefore the evaluation of tumor-related methylated DNA in plasma can be a simple and promising minimally invasive method for early cancer screening, especially using automated isolation techniques. Our aim was to analyse the methylation level alterations of 4 selected genes along the healthy-adenoma-CRC sequence in plasma samples. Moreover, we aimed to test different preservative blood collection tubes and automated cfDNA isolation method to determine whether these factors influence the DNA methylation patterns. Material and methods 121 blood samples were collected in K3EDTA tubes, and manual cfDNA isolation was performed. The methylation pattern of SFRP1 , SFRP2 , SDC2 and PRIMA1 was determined using MethyLight PCR containing multiplex preamplification step. Further 30 blood samples were collected in Roche Cell-Free DNA Collection Tubes, which was developed for stabilisation of cfDNA with preservative reagents. In case of these samples, in addition to manual isolation, MagNA Pure 96 Instrument was used for automated cfDNA extraction. Results and discussions Elevated methylation of the genes was observed in 70% to 90% of adenoma and CRC patients. Average DNA methylation level was SFRP1, SFRP2, SDC2 and PRIMA1 was observed with methylation percentage values of 21.77%±33.32%; 6.82%±17.1%; 12.06%±24.37% and 13.66%±25.14%. Using combined analysis, the 4 markers were able to distinguish CRC patients from normal with 91.5% sensitivity and 97.3% specificity and could differentiate AD samples from controls with 89.2% sensitivity and 86.5% specificity. Interestingly, Roche blood collection tube and automated cfDNA isolation method influence the detected methylation levels; however, methylation differences still have been found between the clinical groups, especially in case of PRIMA1 and SFRP1 genes. Conclusion Our findings suggest that the 4 markers can be promising epigenetic biomarkers for colorectal adenoma and CRC detection with high sensitivity. Automated isolation procedures may contribute to wide-spread, screening applications of methylation markers; however, further developments are needed.

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic-Based Multiplex PCR and Next-Generation Sequencing

Circulating tumor DNA (ctDNA) has potential to serve as a biomarker for noninvasive monitoring of treatment response and disease progression. However, broad clinical applicability of ctDNA has been limited by the low sensitivity, throughput, and patient coverage offered by existing ctDNA detection methods. Herein, we report the adaptation and characterization of the microfluidics multiplex PCR sequencing technology for high-throughput and sensitive quantitation of ctDNA. A multiplex PCR preamplification step was developed and incorporated into the microfluidics multiplex PCR sequencing work flow to enable low-input ctDNA analysis with enhanced sensitivity. An empirical bayesian model was developed to characterize both position and substitution-associated system errors specific to this platform and provided a tailored approach to greatly enhance the confidence and accuracy of variant calling for ctDNA analysis. Clinical validation of this platform for ctDNA mutation detection demonstrated an overall sensitivity of 92% and specificity of 100% when using mutation calls in the matched tumor tissues as a benchmark. Finally, we established an early proof of concept of clinical utility of this ctDNA work flow for monitoring disease progression using clinical trial samples. Our novel ctDNA work flow provides a high-throughput and sensitive platform that can be implemented in clinical trials for mutation detection and disease monitoring from plasma ctDNA.

Detection of trace amounts of target DNA from massive background of nucleic acids by using the LM-PCR-based preamplification method.

The sensitivity and specificity of DNA detection may decrease when the target DNA is in very low abundance. To effectively detect trace amounts of target DNA from massive background of nucleic acids, we have developed a powerful multiplex preamplification method based on ligation-mediated PCR that can greatly enrich multiple target DNAs from massive backgrounds. By employing type IIS restriction endonuclease (REase) and specifically designed oligonucleotide adapters, target DNA can be preamplified with high efficiency and sensitivity. Combining with normal PCR, 10 copies of target DNA was effectively detected from over 108 times more excessive backgrounds with high specificity and 10 times more effectively than conventional PCR. In particular, the usage of universal primer in the preamplification PCR (pre-amp PCR) ensured that multiple targets could be equivalently amplified, which was confirmed by quantitative PCR (qPCR), indicating it could meet the demands of high-throughput detection. The flexibility and applicability of pre-amp PCR was validated by using different microorganisms DNA as targets and employing two different type IIS REases. The results suggest that the pre-amp PCR method has broad application prospects in various gene detection fields.

Abstract 2740: High-throughput and sensitive quantification of circulating tumor DNA

Abstract Circulating tumor DNA (ctDNA) has a great potential to serve as non-invasive biomarkers for monitoring disease progression and treatment response. However, its broad clinical applications have been hindered by sensitivity, throughput and complexity of existing ctDNA detection methods. Here we report an adaptation of a multiplex PCR pre-amplification (PreAmp) into the microfluidic multiplex PCR (MMP-Seq) technology for high-throughput and sensitive quantitation of ctDNA. We have demonstrated that PreAmp MMP-Seq enabled sensitive (down to 0.6% allele frequency), reproducible (R2=0.97), accurate and quantitative mutation detection from as low as 1.6 ng DNA input. An Empirical Bayesian Model has been established to estimate background error rates for every base of amplicons in the ctDNA panel using data from 43 normal plasmas. 0.1-0.3% of median error rates were observed across all positions. 149 longitudinal plasma samples from 12 ovarian and 10 pancreatic cancer patients of PhI DMUC5754A anti-MUC16 ADC trials were then profiled using PreAmp MMP-Seq. 79% (11/14) driver mutations found in tissue samples were also identified in matching baseline plasma samples at frequencies well above estimated error rates. Moreover, we found that changes of corresponding ctDNA concentration in post-treatment plasma samples were correlated with changes in tumor burdens and circulating protein marker levels. In conclusion, the MMP-seq PreAmp workflow provides an inexpensive, reproducible and flexible solution for mutation detection in plasma DNA samples. Citation Format: Yinghui Guan, Oleg Mayba, Shan Lu, Thomas Sandmann, Younjeong Choi, Walter Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W. Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Yulei Wang. High-throughput and sensitive quantification of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2740. doi:10.1158/1538-7445.AM2017-2740

AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A

BackgroundTo develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. MethodsThe design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. ResultsPLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. ConclusionsAccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.

Quantitative large scale gene expression profiling from human stem cell culture micro samples using multiplex pre-amplification

Transcriptional profiling is a powerful tool to study biological mechanisms during stem cell differentiation and reprogramming. Genome-wide methods like microarrays or next generation sequencing are expensive, time consuming, and require special equipment and bioinformatics expertise. Quantitative RT-PCR remains one of today’s most widely accepted and used methods for analyzing gene expression in biological samples. However, limitations in the amount of starting materials often hinder the quantity and quality of information that could be obtained from a given sample. Here, we present a fast 4-step workflow allowing direct, column-free RNA isolation from limited human pluripotent stem cell (hPSC) cultures that is directly compatible with subsequent reverse transcription, target specific multiplex pre-amplification, and standard SYBR-Green quantitative PCR (qPCR) analysis. The workflow delivers excellent correlations in normalized gene-expression data obtained from different samples of hPSCs over a wide range of cell numbers (500–50,000 cells). We demonstrate accurate and unbiased target gene quantification in limiting stem cell cultures which allows for monitoring embryoid body differentiation and induced pluripotent stem cell (iPSC) reprogramming. This method highlights a rapid and cost effective screening process, allowing reduction of culture formats and increase of processing throughputs for various stem cell applications.

Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.

Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched. Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%-0.1% mutation abundance by use of Ion-Torrent and 0.1%-0.3% by use of Sanger sequencing. Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation.

Diagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic "LabDisk" system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 μL serum sample. The turnaround time of the complete analysis from "sample-to-result" was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.

Validation of a multiplex reverse transcription and pre-amplification method using TaqMan(®) MicroRNA assays.

Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan® technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.

Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting (FACS)

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence-activated cell sorting (FACS)-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5-6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as five Fos-positive neurons. Methamphetamine induced 3-20-fold increases of immediate early genes arc, homer-2, c-fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not Fos-negative neurons. Immediate early gene mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine-induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.