Establishment and evaluation of direct assay for plasma microRNAs without RNA extraction by quantitative PCR

Abstract

Objective To establish a new method for the detection of plasma miRNAs by real-time fluorescence quantitative PCR without RNA extraction. Methods 7.5 μl plasma separated from normal human blood was treated with equal amount of buffer containing Tween 20, which was used directly as sample for the following reverse transcription and PCR reaction to establish a direct assay for plasma microRNAs. U6 snRNA, miR-24-1-5p and miR-4634 were selected as target microRNAs for the determination of RT-qPCR. Both specificity and amplification efficiency were assessed. The difference of Cq values between traditional quantitative PCR detection method and our new method was also observed. Furthermore, the results of mixed primer or individual primer by RT-qPCR were compared in new established method. Results The amplification products of U6, miR-24-1-5p and miR-4634 show a single melting peak at 81 ℃, 83 ℃ and 84.2 ℃, respectively, without primer dimer peak or non-specific peak in all 8 cases of healthy human plasma samples. Amplification efficiency for miR-24-1-5p reached 1.1 detected in plasma direct assay. The amplification curve of U6, miR-24-1-5p and miR-4634 is parallel in each sample, which demonstrated that the amplification of slope is consistent. The Cq value of miR-4634 determined by direct assay is higher than the result of the method by RNA extraction（plasma direct assay 30.98±0.17，RNA extraction 29.22±0.21；t=8.475，P 0.05）and miR-24-1-5p(plasma direct assay 20.73±0.14，RNA extraction 21.06±0.21；t=1.749, P>0.05) although they had significant correlation（r=0.860 5,0.728 9,0.748 5,P<0.05）. No significant difference of Cq value between mixed primer reverse transcription and individual primer reverse transcription. Conclusion A novel RT-qPCR direct assay of plasma miRNAs is established, which can improve the efficiency of plasma miRNAs determination in potential clinical applications. （Chin J Lab Med，2014,37：119-122） Key words: MicroRNAs; Real-time polymerase chain reaction; Plasma