[Study on the Sample Preservation Temperature and Period in Circulating MicroRNA Quantification Using Spike-In Control].

MicroRNA in body fluid is called circulating microRNA and is expected to be a non-invasive stable biomarker of various diseases. In real-time RT-PCR of circulating microRNA, synthetic non-human microRNA, such as cel-miR-39, is used as spike-in control RNA instead of endogenous control RNA. Spike-in control RNA, which is added into sera or plasma just before RNA extraction, does not reflect microRNA degradation in the period between blood sampling and RNA extraction. Therefore, it is essential to prevent degradation of circulating microRNA in this period for better reproducibility of the quantification of microRNA using spike-in control. To address this issue, we analyzed the stability of cel-miR-39 on ice and circulating miR-21 and miR-223 in whole blood and serum. The synthetic cel-miR-39 in RNase-free water was stable for at least 3 hours on ice. Degradation of miR-21 and miR-223 in whole blood was not observed for 3 hours at room temperature. However, miR-223 in serum was apparently degraded within 24 hours at 4°C and the stability levels of miR-21 and miR-223 in serum were significantly different (fold changes of miR-21 and miR- 223 within 24 hours were 0.891 and 0.485, respectively). These results show that it is essential to avoid long-term storage of sera at 4°C to prevent degradation of microRNA in the quantification of circulating microRNA using spike-in control.