Use of chip-based PCR for 3D absolute digital quantification of microRNAs molecules for the non-invasive diagnostic screening of human colon cancer in stool

Abstract

Received: April 07, 2018; Accepted: April 24, 2018; Published: April 27, 2018 There is no validated approach to screen for colon cancer (CC) quantitativley on the marke, using molecular sensitive approach today, 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 obstacles for developing a sensitive, economical and a non-invasive molecular colon cancer screening test, we have first carried out a microarray miRNA qualitative study, using Affymetrix GeneChip miRNA 2.0 Arrays, on immunocaptured and eniched 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 Up-Regulated, miR-19a, miR-20a, miR-21, miR-31, miR34a, miR-96, miR-106a, miR-133a, miR-135b, miR-206, miR-224 and miR-302; and 2 Down-Regulated, miR-143 and miR-145). This was followed by an absolute quantitative digital PCR on these stool samples from the same stool samples. In which 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, which was measured using a chip-based Absolute QuantStudio 3D Digital PCR analysis, to validate microarray results. To guarentee that we have used human and not bacterial small total RNA, we have carried out coextraction protocols with E. coli K1 strain RS18, compared Agilent electrophoretic patterns, and also sequenced random samples, using mRNA/miRNA sequencing.