MicroRNA Isolation by Trizol-Based Method and Its Stability in Stored Serum and cDNA Derivatives

摘要

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Since aberrant expression of miRNAs has been proposed as usage for blood-based biomarkers, hence reliable techniques for miRNA isolation as well as stability of miRNAs in various stored conditions needs to be explored. This present study aimed to investigate the efficacy of the Trizol-based isolation technique and the stability of miRNAs in stored serum and cDNA derivatives. Total RNA, including miRNAs, was isolated from human serum and a comparison of the efficiency of the Trizol LS reagent isolation method against the miRNeasy mini kit was conducted. Expression of RNU6, miR-145, and miR-20a was determined by quantitative real-time polymerase chain reaction (qRT-PCR). We showed that Trizol LS isolation yielded significantly lower RNA concentrations than that of the miRNeasy mini kit by approximately 35%. Purity of the isolated RNAs by both methods was similar. RNU6, miR-145, and miR-20a degraded at room temperature, but all genes were stable at 4ºC, -20ºC and -80ºC for a 72-hrs period, in both serum and cDNA storage conditions. In the stored cDNA derivatives, we observed the stability of RNU6, miR-145, and miR-20a for 3 months at -20ºC, and all genes also resisted 4 repeated freeze-thaw cycles at -20ºC. In conclusion, the Trizol-based method is efficient as well as economical to use for quantification of circulating miRNAs. In addition, we proposed that the storage of miRNA-derived cDNAs may be an alternative choice to avoid the stability effect.