Factors influencing degradation kinetics of mRNAs and half-lives of microRNAs, circRNAs, lncRNAs in blood in vitro using quantitative PCR

Abstract

RNAs are rapidly degraded in samples and during collection, processing and testing. In this study, we used the same method to explore the half-lives of different RNAs and the influencing factors, and compared the degradation kinetics and characteristics of different RNAs in whole blood and experimental samples. Fresh anticoagulant blood samples were incubated at room temperature for different durations, RNAs were extracted, and genes, including internal references, were amplified by real-time quantitative PCR. A linear half-life model was established according to cycle threshold (Ct) values. The effects of experimental operations on RNA degradation before and after RNA extraction were explored. Quantitative analysis of mRNA degradation in samples and during experimental processes were explored using an orthogonal experimental design. The storage duration of blood samples at room temperature had the greatest influence on RNA degradation. The half-lives of messenger RNAs (mRNAs) was 16.4 h. The half-lives of circular RNAs (circRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were 24.56 ± 5.2 h, 17.46 ± 3.0 h and 16.42 ± 4.2 h, respectively. RNA degradation occurred mainly in blood samples. The half-life of mRNAs was the shortest among the four kinds of RNAs. Quantitative experiments related to mRNAs should be completed within 2 h. The half-lives of circRNAs and lncRNAs were longer than those of the former two.

Figure 1

Linear detection using a high-efficiency blood RNA extraction kit (A). Linear detection by microspectrophotometry (B). Linear detection by real-time quantitative PCR amplification (C).

Figure 2

Dynamic degradation curves for mRNA, circRNAs, lncRNAs and miRNAs in blood after natural logarithm transformation.