Objective

Circulating miRNAs have been extensively used in studies of neurological diseases. Thus, methods to extract high quantity total RNA for RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) are needed. However, the extraction of sufficient high-quality nucleic acids from circulating blood is difficult. Differences in eccentricity, cryopreservation conditions and extraction methods may affect RNA quantity and quality. Here, we systematically compared six blood-RNA extraction protocols (protocols 1, 2, 3, 4, 5, and 6; see the methods section for details).

Results

Protocol 1 yielded the highest quality and quantity of RNA; protocol 2, protocol 5 and protocol 6 produced RNA of intermediate quality; and protocols 3 and 4 yielded the lowest quality RNA. The RNA integrity number (RIN) for isolated RNA was > 9.0 when protocol 1 or protocol 2 was used, > 8.0 when protocol 5 was used, and > 7.0 when protocol 6 was used; lower values were obtained when protocol 3 or 4 was used. The RNA extracted from circulating blood using protocol 1 was most intact and suitable for RT-qPCR and RNA-seq.

Conclusions

The quality of RNA extracted from circulating blood is affected by high-speed centrifugation and cryopreservation. Adding an RNA stabilizer during the cryopreservation of circulating blood significantly improved RNA quality and quantity. The quality of extracted RNA from circulating blood is improved when using TRIzol relative to that attained with a commercial kit.

中文翻译：

---

从循环血中制备 RNA 用于 RNA 测序的不同方案的比较

客观的

循环 miRNA 已广泛用于神经系统疾病的研究。因此，需要为 RNA 测序 (RNA-seq) 和实时定量聚合酶链反应 (RT-qPCR) 提取大量总 RNA 的方法。然而，从循环血液中提取足够的高质量核酸是困难的。离心率、冷冻保存条件和提取方法的差异可能会影响 RNA 的数量和质量。在这里，我们系统地比较了六种血液 RNA 提取方案（方案 1、2、3、4、5 和 6；详见方法部分）。

结果

方案 1 产生了最高质量和数量的 RNA；方案 2、方案 5 和方案 6 产生了中等质量的 RNA；方案 3 和 4 产生的 RNA 质量最低。使用方案 1 或方案 2 时，分离 RNA 的 RNA 完整性数 (RIN) > 9.0，使用方案 5 时 > 8.0，使用方案 6 时 > 7.0；使用协议 3 或 4 时获得的值较低。使用方案 1 从循环血液中提取的 RNA 最完整，适用于 RT-qPCR 和 RNA-seq。

结论

从循环血液中提取的 RNA 的质量受高速离心和冷冻保存的影响。在循环血液的冷冻保存过程中添加 RNA 稳定剂可显着提高 RNA 的质量和数量。相对于商业试剂盒，使用 TRIzol 从循环血液中提取的 RNA 的质量有所提高。