Comparison of different protocols of RNA preparation from circulating blood for RNA sequencing

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).

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.

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.