dPCR System Research Articles

Abstract 5316: Quantitative cancer analysis using digital PCR: Absolute counting of DNA (solid tumors and liquid biopsies in glioma, breast, and colon cancer) and RNA (mRNA and miRNA using One-Step RT-dPCR)

Abstract Background: There is a need for more accurate and sensitive methods to quantify nucleic acids present in precious samples and non-invasively collected “liquid biopsy” material. Digital PCR (dPCR) is one technology with recent advances in automation, enabling researchers to sensitively quantify a sample's nucleic acids by adding up digital fluorescent counts of single target molecules. Particularly exciting are the emerging uses of cell-free nucleic acids found in peripheral body fluids for dynamic measurements of total body mutational burden of heterogeneous cancers, potentially reducing the need for multiple invasive tumor biopsies. In addition, combined measurement of mRNA and miRNA RNA biomarkers from a single sample, for example from cell-free exosomes, is of clinical interest. Here we present results from several studies using dPCR for DNA and cDNA molecule counting with various cancers, as well as in a first time demonstration quantifying RNA directly with multiple miRNA species, and mRNA together with miRNA. Method & Results: Picoliter droplet digital PCR was used to perform single molecule counting. Standard qPCR assay reagents for each target were combined in duplex or multiplex formats together with samples, and partitioned into millions of 5 picoliter-sized droplets on a RainDance RainDropTM dPCR system. Each sample was then thermal cycled prior to digital fluorescent quantification. In separate studies we show quantification of mutant IDH1 mRNA (cDNA converted) in glioma patient cerebrospinal fluid extracellular vesicles, PIK3CA mutations and methylation of CyclinB2 and Retinoic Acid Receptor promoters in breast tumor DNA, KRAS and BRAF mutations in colorectal cancer plasma cell-free DNA, and multiplexed miRNA biomarkers from plasma (cDNA converted). In addition, we show direct quantification of mRNA molecules in highly precise multiplex One-Step RT-dPCR measurements across a wide dynamic range. Finally, we show simultaneous One-Step RT dPCR direct digital counting of miRNA and mRNA molecules. Conclusion: Droplet digital PCR is a highly sensitive and precise approach to quantify nucleic acids without the need for standard curves. With the RainDance RainDrop dPCR platform there are sufficient partitions and compatibility with various robust chemistries to apply dPCR to DNA and RNA analyses from a variety of biofluids and neoplasms, enabling accurate absolute counting of multiple target molecules across a wide dynamic range. Citation Format: Michael L. Samuels, Valerie Taly, Leonora Balaj, Saumya Das, Ben Ho Park. Quantitative cancer analysis using digital PCR: Absolute counting of DNA (solid tumors and liquid biopsies in glioma, breast, and colon cancer) and RNA (mRNA and miRNA using One-Step RT-dPCR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5316. doi:10.1158/1538-7445.AM2014-5316

Abstract 808: Quantitative digital PCR analysis of cancer gene promoter methylation using low amounts of input DNA.

Abstract Silencing of tumor suppressor and tumor-related gene transcription by hypermethylation at promoter CpG motifs is a significant mechanism at work in human tumorgenesis. Various methods have been developed to analyze DNA methylation levels, both across the genome and at specific loci, in order to discover and interrogate disease relevant loci for methylation-based transcriptional control. Methods able to quantitatively measure differences in DNA methylation between normal and cancer cells provide promising sources for biomarker identification and assessment. Here we report on the use of a methylation assay (Zymo Research's OneStep qMethyl™) and digital PCR counting (RainDrop™ dPCR) to enable quantitative measurements of DNA methylation at specific genomic loci using small amounts of non-bisulfite converted DNA. MGMT promoter methylation measurements are used to illustrate the method's robust quantification of small percent methylation changes using low amounts of input DNA. Zymo Research's OneStep qMethyl™ Kit is used for the detection of region-specific DNA methylation via the selective amplification of methylated cytosines after digestion with methylation-sensitive restriction enzymes. RainDance Technologies’ RainDrop™ dPCR system is a highly sensitive tool for precise quantification of nucleic acids using probe-based qPCR reagents. RainDrop offers unique analytical advantages for clinical diagnostics due to its exceptionally high sensitivity for absolute quantification and capability to multiplex assays, using a wide dynamic range of input DNA in a contamination-free and simple workflow. The combination of both technologies enables a novel, fast, and robust method for quantitative methylation analysis of small amounts of DNA. The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes mutagenic alkyl adducts from DNA, and its epigenetic silencing by promoter methylation confers susceptibility to DNA-damaging alkylating agents in glioblastomas, melanomas, and lung cancer cell lines, and is increased in cancers including advanced gastric carcinomas, colorectal tumors, and triple-negative breast cancers. A sensitive, quick, and cost-effective quantitative method for assessing MGMT promoter methylation would greatly enhance the diagnostic potential for this candidate cancer biomarker. Here we show digital quantification of MGMT promoter methylation across the range from 0 to 100%, demonstrating a linear correlation that enables less than 15% methylation changes to be robustly measured using less than 20 nanograms of input DNA. This method provides a sensitive count of region-specific methylation percentages using an easy workflow. Continuing work will make use of RainDrop's unique ability to multiplex assays to enable combining MGMT methylation analysis with additional cancer relevant methylation biomarkers in a single assay. Citation Format: Michael L. Samuels, Ryan Kemp, Frances Long, Jill Petrisko, Lam Nguyen, Manuel Krispin. Quantitative digital PCR analysis of cancer gene promoter methylation using low amounts of input DNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2013-808