Direct S-Poly(T) Plus assay in quantification of microRNAs without RNA extraction and its implications in colorectal cancer biomarker studies

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 the lack of simple but robust miRNA measurements. Methods We built up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting miRNAs without RNA purification. In this study, the method was optimized and compared with other RNA purification-based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, seven potential miRNA biomarkers of colorectal cancer were validated. Results It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20 μl) and time (~ 140 min) with this approach. The sensitivity of this method was 2.7–343-fold higher than that of the 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 the area under receiver operating characteristic (ROC) curve ranging from 0.79 to 0.94 (p value < 0.001). Conclusions This simple and robust protocol may have strong impact on the development of specific miRNAs as biomarkers in clinic.

Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification

“…To perform library preparation for highly fragmented cfRNAs, we optimized the S-Poly(T) Plus method that was previously developed for high-throughput small RNA profiling 28 – 30 . In brief, the input cfRNA was subjected to 3’-end polyadenylations along with reverse transcription (RT) in a one-step reaction (Fig.…”

SA-ODG platform: a semi-automated and PCR-free method to analyse microRNAs in solid tissues

“…8 Yet, miRNAs present several challenges due to their short sequence, difficulties in the normalization analysis and, in the case of those found in biofluids, their low abundance. Several variances of the RT-qPCR standard procedure have been introduced such as ddPCR, 42 RT-qPCR-DS (direct detection in serum), 43 Direct S-Poly(T) Plus 44 and multiplex circulating Fireplex 16 to avoid RNA extraction or retro transcription steps.…”

miRNA-Based Potential Biomarkers and New Molecular Insights in Ulcerative Colitis

“…On the other hand, there is no uniform quantitative standard for the detection of miRNA, which is one of the reasons limiting the development of miRNA biomarkers. Especially for complex samples, such as feces, there is a need for more effective and standardized measurement method (Niu et al, 2019). Measuring exosomal-derived miRNAs in blood may reduce the interference of other components, but the technology and cost of isolating exosomes are also challenging issues (Vasconcelos et al, 2019).…”