Aiso Toshiko, Sekine Nariko, Takagi Yasushi, Ohnishi Hiroaki
Rinsho Byori. 2015 Jun;63(6):688-93.
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.
体液中的微小RNA被称为循环微小RNA，有望成为各种疾病的非侵入性稳定生物标志物。在循环微小RNA的实时逆转录聚合酶链反应（RT-PCR）中，合成的非人类微小RNA，如cel-miR-39，被用作加标对照RNA，而不是内参RNA。在RNA提取前加入血清或血浆中的加标对照RNA，并不能反映采血和RNA提取期间微小RNA的降解情况。因此，为了使用加标对照更好地重现微小RNA定量结果，在这一期间防止循环微小RNA降解至关重要。为解决这一问题，我们分析了cel-miR-39在冰上的稳定性以及全血和血清中循环miR-21和miR-223的稳定性。无核糖核酸酶水中的合成cel-miR-39在冰上至少3小时内稳定。室温下3小时未观察到全血中miR-21和miR-223的降解。然而，血清中的miR-223在4℃下24小时内明显降解，血清中miR-21和miR-223的稳定性水平存在显著差异（24小时内miR-21和miR-223的倍数变化分别为0.891和0.485）。这些结果表明，在使用加标对照定量循环微小RNA时，必须避免血清在4℃下长期保存以防止微小RNA降解。
