Quantification of MicroRNAs for the Diagnostic Screening of Colon Cancer in Human Stool by Absolute Digital(d)PCR*

Farid E. Ahmed¹*, Mostafa M Gouda^2,3, and Nancy C. Ahmed¹

Received: January 08, 2019   Published: January 18, 2019

DOI: 10.32474/SCSOAJ.2019.01.000125

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Abstract

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). By employing earlier on 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  1 cm, villous or tubvillous, or with high grade dysplasia), three stage 2, three stage 3, and three stage 4] in triplicates, this allowed for selection of 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). Then carrying out an absolute quantitative digital PCR on these 15 stool samples from TNM stages 0-4 on total small RNA extracted by immunocapture, followed by RT that employed a Custom TaqMan® miRNA Reverse Transcription (RT) Kit and TaqMan RT Primer Pool, and absolute quantification of miRNAs, in copies/μl, measured using a chip-based Absolute QuantStudio 3D Digital PCR analysis, allowed for validating the microarray results. To ensure that human and not bacterial small total RNA was chosen, coextraction protocols with E. coli K1 strain RS18 was carried out, followed by comparing Agilent electrophoretic patterns with human and bacterial electrophoretic patterns, and also random samples were sequenced using mRNA/miRNA sequencing, to ensure that human and not bacterial mRNA was chosen.

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