Absolute Quantification of Colon Cancer MicroRNAs with 3D Digital, Chip-Based PCR

There is currently no validated micro(mi)RNA diagnostic stool test to screen for colon cancer (CC) on the market because of the complexity of fecal density, vulnerability of stool to daily changes, and the presence of three sources of miRNAs in stool (cell-free from fecal homogenates, exsosomal miRNAs from fecal exosomes, and fecal colonocytes).To address these complexities, we have earlier on carried out a microarray miRNA experiment, using Affymetrix Gene Chip miRNA 2.0 Arrays, on immunocaptured and enriched stool colonocytes of 15 subjects [three healthy controls and twelve colon cancer patients [three TNM stage 0-1 (e.g., polyps ≥ 1 cm, villous or tubvillous, or with high grade dysplasia), three stage 2, three stage 3, and three stage 4] in triplicates to select a smaller panel of 14 preferentially expressed mature miRNAs associated with colon cancer (12 .In a subsequent preliminary study, absolute quantitative digital PCR on these 15 stool samples from stages 0-4 was subsequently carried out on total small RNA extracted by immunocapture, followed by RT that employed TaqMan® miRNA Reverse Transcription (RT) Kit and a Custom TaqMan RT Primer Pool, and absolute quantification of miRNAs, in copies/µl, was measured using a chip-based Absolute QuantStudio 3D Digital PCR analysis, to validate microarray results.To ensure that we have chosen human and not bacterial small total RNA, we have carried out coextraction protocols with E. coli K1 strain RS18, compare Agilent electrophoretic patterns, and also sequenced random samples throughout this research using mRNA/miRNA sequencing.Our initial quantitative dPCR miRNA data presented in this article, showed that the quantitative changes in the expression of a few mature miRNA genes in stool, which are associated with right and left colon cancer, would provide for a more convenient, sensitive and specific diagnostic screening markers.More useful than those test markers currently available on the market, such as the low-sensitivity (<15%) fecal occult blood test (FOBT); result in better compliance; and is more economical than the invasive and expensive colonoscopy exam in colon cancer, which can be cured if that cancer is detected at the early TNM stages, and that becomes incurable and deadly if not diagnosed before metastasis.Initial test performance characteristics of the miRNA approach showed that the test has a high numerical predictive value in colon cancer.Moreover, underpinning of the miRNA markers as a function of total RNA showed that the test can numerically differentiate between control subjects and colon cancer patients, particularly at the early stages of that curable cancer.We propose to extend our initial research results to a larger prospective and randomized five-years nested casecontrol study, to validate the expression of the above 14 miRNAs, in stool of 180 individuals in an epidemiologically designed study, using [30 controls and 150 colon cancer patients [thirty precancerous polyps (stage 0-1), forty five stage 2, and seventy-five colon cancer stages 3 or 4] chosen randomly by an epidemiological method from MiRNA Up-Regulated Down-Regulated Chromosome LocationKnown Putative Cancer Target Gene(s) MiR-19a Yes No 13q31.