Use of RT-qPCR for Measuring the Expression of the Fecal MicroRNA from the Consumption of Pomegranate Fruit Juice

The ongoing process involves the modification and improvement of the stool test that has been validated for the purpose of diagnostic screening of colon cancer (CC). This particular test focuses on the detection of micro(mi)RNAs. The need for this update is primarily driven by the complex nature of fecal density, the variations in stool composition that occur on a daily basis, and the presence of miRNAs from various sources in the stool. These sources include cell-free homogenates, exosomal miRNAs, and stool colonocytes. In a previous study conducted by our team, we performed a microarray miRNA experiment utilizing Affymetrix GeneChip miRNA 2.0 Arrays. The experiment involved the analysis of immunocaptured and enriched stool colonocytes obtained from 15 human subjects. The colon cancer patients were further categorized into different stages, namely TNM stage 0–1, TNM stage 2, TNM stage 3, and TNM stage 4. Each sample was analyzed in triplicates to ensure accuracy and reliability of the results. From this experiment, we successfully identified a panel of 14 mature miRNAs that exhibited preferential expression in association with colon cancer. Out of these miRNAs, 12 were found to be upregulated, specifically 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. On the other hand, two miRNAs, namely miR-143 and miR-145, were found to be downregulated. In order to further validate our findings, we performed absolute quantitative digital (d) PCR on the 15 stool samples, which spanned the different stages of colon cancer (0–4). This analysis involved the extraction of total small RNA through the process of immunocapture, followed by reverse transcription (RT) and absolute quantification of miRNAs. Additionally, throughout the course of our research, we also conducted mRNA/miRNA sequencing on random samples to ensure that the selected RNAs were indeed of human origin and not of bacterial origin.