Quantification of Micrornas by Absolute Dpcr for the Diagnostic Screening of Colon Cancer

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 first carried out a microarray miRNA experiment, using Affymetrix GeneChip 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◻ ³ 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 Up-Regulated, miR-19a, miR-20a, miR-21, miR-31, miR-34a, miR-96, miR-106a, miR-133a, miR-135b, miR-206, miR-224 and miR-302; and 2 Down-Regulated, miR-143 and miR-145). In a subsequent validation study 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, absolute quantification of miRNAs, in copies/µl, was measured using a chip-based Absolute QuantStudio 3D Digital PCR analysis. 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 herein showe 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 thatare 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.