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)
Authors: Samuels Michael L. , Taly Valerie , Balaj Leonora , Das Saumya , Park Ben H. 
Published: May 2015

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