Abstract
Background: Advances in microRNAs (miRNAs) biomarkers have generated disease markers with potential clinical values, however, none of these published results have been applied in clinic until today. The main reason could be lacking of simple but robust miRNA measurements. Methods: We build up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting of miRNAs without RNA purification. In the study, this method was optimized and compared with other purification-RNA based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, 7 potential miRNA biomarkers of colorectal cancer were validated. Finding: It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20ul) and time (~140 minutes) with this approach. The sensitivity of this method was 2.7~343-fold higher than that of stem-loop method, and comparable with S-Poly(T) plus method. 7 validated miRNA biomarkers of colorectal cancer by Direct S-Poly(T) plus assay could discriminate colorectal cancer Stage I from healthy individuals, and promised satisfactory discrimination with area under ROC curve ranging from 0.79 to 0.94 (p value<0.001). Interpretation: The sensitivity of Direct S-Poly(T) Plus rely on: a complete denaturation of miRNA-containing protein complexes and endogenous RNase; an elaborately designed RT primer; a single-step reaction containing polyadenylation and reverse transcription and a high-activity DNA polymerase of thermus aquaticus (Taq) for the crude cDNA. Funding: National Natural Science Foundation, Shenzhen Municipal Basic Research Program, Shenzhen University start-up funding and Shenzhen University Interdisciplinary Innovation Team Project. Declaration of Interest: All authors express no conflict of interest and have nothing to disclose. Ethical Approval: Institutional Ethics Committee at the Shenzhen People’s Hospital (Shenzhen, China) approved the experiments, and written informed consent was obtained from all subjects. All procedures were performed according to the approved guidelines.
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