Highly specific real-time quantification of diverse microRNAs in human samples using universal primer set frame

Abstract

In this study, one group of universal primer set frame, composed by one reverse transcription (RT) primer frame and a pair of quantitative real-time polymerase chain reaction (qRT-PCR) primer frames, was elaborately screened and designed by homebuilt software for sensitive and specific quantification of diverse miRNAs. The universal primer set frame can be applied for multiplex miRNAs detection by simply changing the RT-X part of RT primer frame and RP-Y part of qRT-PCR reverse primer frame based on target sequence. The maximum similarity of RT-Y, RT-Z and qRT-PCR forward primer to the human genome and human transcriptome is less than 76%, ensuring the high specificity in human sample detection. The high sensitivity and broad dynamic linear range of the developed approaches by using designed primer set frame were demonstrated on the in vitro detection of miR-21 and miR-155, with dynamic range of 10 fM to 10 nM and detection limit of 3.74 × 10−15 M and 5.81 × 10−15 M for miR-21 and miR-155, respectively. In particular, the developed assays also have high sequence specificity which could clearly discriminate a single base difference in miRNA sequence. The contents of miR-21 and miR-155 in tissue and serum samples have been successfully detected using the developed assays. Results indicated that miR-21 and miR-155 were elevated in cancer tissue and serum specimens than that of normal samples, implying the developed assays hold a great promise for further application in biomedical research and early clinical diagnosis. More importantly, the primer set frame can be universally used in any miRNA investigation.