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MicroRNAs (miRNAs) are small non-coding RNAs that function to regulate gene expression at transcriptional and post-transcriptional level. Mature miRNAs are short, usually 18-23 nucleotides (nt) in length, with high homology in the seed region (position 2-8 from the 5′ end of a mature miRNA) within each family. This presents a great challenge when designing assays for quantitation by real-time polymerase chain reaction (PCR). Existing miRNA assays often trade in sensitivity and/or assay performance for specificity. Comparison between two different mature miRNAs with quantitative real-time PCR (qPCR) requires generating standard curves and characterizing PCR efficiency and sensitivity. Here we report a chip-based digital PCR technology. With this technology, we are able to accurately quantitate mature miRNAs without characterizing and optimizing the assay. Briefly, we input the same amount (ratio=1:1) of synthetic hsa-miR-19b and hsa-miR-92a. We then quantitated the miRNAs using reverse transcription followed by quantitative RT-PCR or chip-based QuantStudio™ 3D digital PCR (dPCR). Data from qPCR showed a delta Ct=2.58 between the cDNAs. This converted to a 3.84 fold difference between the two mature miRNAs, which deviated 284% from the input ratio, which equals 1. However, the data from dPCR indicated that the ratio between the amounts of the two mature miRNAs was 0.94 ± 0.02 (SD). Although statistically different, the results from dPCR deviated only 5% from the input ratio. Compared to qPCR, dPCR significantly enhanced the precision in sample quantitation. In summary, our study demonstrated that the chip-based dPCR technology has greatly increased precision in quantitating miRNA samples. The simplified workflow and improved output of dPCR are very useful features in broad applications such as revealing small changes in the miRNA expression levels. Citation Format: Yalei Wu, Victoria Dahlhoff, Lin He, Caifu Chen. Accurate quantitation of micro RNA by chip-based digital PCR. [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 5241. doi:10.1158/1538-7445.AM2014-5241

[OBJECTIVES] Genetic alternations of EGFR and KRAS are frequently found in lung cancer cells. Accurate detection of these mutations from lung cancer peripheral blood may provide a noninvasive means for early cancer detection and disease monitoring. The goal of this study is to develop a quantitative analysis of EGFR and KRAS mutations both of ctDNA in plasma and of CTCs in blood cells from lung cancers by digital castPCR (competitive allele specific TaqMan-base PCR) technology. [METHODS] Molecular analysis and enumeration of CTCs in peripheral blood cells were analyzed by combining sample partition and digital castRT-qPCR assays without prior biophysical processing. The castPCR can detect rare copies of mutant alleles with a 6-log dynamic range and < 5-copy detection sensitivity for EGFR and KRAS mutations. Quantitative analysis of EGFR and KRAS mutations in ctDNA was done in OpenArray platform by digital castPCR. For CTC detection, whole blood samples with spiked-in known mutation and gen marker lung cancer cell lines were partitioned onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none together with normal white blood cells and red blood cells. RNAs and/or DNAs were extracted by magnetic beads and were pre-amplified prior to molecular detection. Genetic mutations and cell type specific markers (CK19) for CTC identification and enumeration were determined by castRT-qPCR and TaqMan Gene expression (GEx) assays, respectively. Blood samples from lung cancer patients were first separated into blood cells and plasma portions before CTC detections. The sample plasma portions were used to detect EGFR and KRAS mutations of ctDNA by digital castPCR in OpenArray platform. [RESULTS] The sample partition process resulted in a relative CTC enrichment or digital enrichment of 20 - 400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. The RT-castPCR and TaqMan GEx assays accurately quantify the number of spiked-in tumor cells (10 - 60 cells/mL) in whole blood with known EGFR and/or KRAS mutations and CK19. For 10 blood samples from lung cancer patients, CTCs were detected from all patients even with stage I and II of 3 patients. In the plasma portions of the same samples, digital castPCR by OpenArray was able to detect EGFR mutations (p.L858R) and 19 mutations of EFGR exon 9. The results of EGFR mutations are consistent between intact CTCs and ctDNA. [CONCLUSION] Our preliminary data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in blood cells without prior biophysical sample enrichment. Digital castPCR by OpenArray can be used to assess circulating tumor DNA. Further test in larger clinical samples are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1736. doi:1538-7445.AM2012-1736