dpcr for the absolute diagnostic quantification of mirnas in human colon cancer stool

Currently, there has been no validated miRNA stool test for the diagnostic detection of colon cancer (CC). That is due to the complexity of fecal density, constant changes in stool every day, and the fact that there are more than one sources of miRNAs in stool (cell-free homogenates, exsosomal miRNAs, and stool colonocytes). Our previous research on immunocaptured and enriched stool colonocytes on 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, employe an Affymetrix GeneChip miRNA 2.0 Arrays containing hundreds of microRNAs, and we selected 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). We then followed up by carrying out an absolute quantitative digital PCR on the above fifteen stool samples from stages 0-4, using total small RNA that was extracted by immunocapture, followed by reverse transcription (RT), and finally using an absolute quantification of the miRNAs, using a chip-based Absolute QuantStudio 3D Digital PCR system, which accurately and quantitativley measured miRNAs in copies/µl. Moreover, to ascertain that we have used human and not bacterial small total RNA, we have also simultaneously performed coextraction of the small total RNA from all samples with an Escherichia coli K1 strain RS18, Agilent electrophoretic patterns, and also sequenced random samples throughout our studies, using mRNA/miRNA sequencing,.
We present in this article our quantitative absolute dPCR miRNA data, showing that the quantitative changes in the expression of a few mature miRNA genes in human stool, associated with right and left colon cancer, provides very sensitive and specific diagnostic miRNA screening markers, which are more useful than those currently available on the market, such as the low-sensitivity (<15%) fecal occult blood test (FOBT); leading to better compliance, and a cheaper test than the invasive and expensive colonoscopy examination. Initial test performance characteristics of the new miRNA approach, showed that the new test has a high numerical predictive value in colon cancer. Underpinning of the miRNA markers as a function of total RNA has shown that this new test numerically and accurately differentiates between control subjects and colon cancer patients at the early stages of that common human cancer, worldwide.