Effects of Freezing and Rewarming Methods on RNA Quality of Blood Samples

RNA extracted from human blood has been widely applied to biological, medical, and clinical research of numerous diseases. Previous studies have demonstrated that high-quality RNA is indispensable to guarantee the reliability of downstream assays. In this study, we investigated the effects of freezing procedures, rewarming methods, and blood components on RNA quality of blood samples. Rabbit blood samples were divided into two groups: (1) whole blood (WB) and (2) blood cell components (BCC) with plasma removed. Samples were frozen using four representative freezing procedures (snap freezing in liquid nitrogen, snap freezing at -80°C, traditional slow freezing, and programmable controlled rate freezing) and rewarmed by placing at 4°C or by vortexing. RNA was extracted using the phenol-chloroform RNA extraction method and measured by an Agilent bioanalyzer. Then, human blood was used to verify the best protocol obtained from the rabbit blood experiment. For the four freezing procedures, there were no differences in RNA integrity. For different rewarming methods, RNA integrity number (RIN) values of RNA extracted from frozen WB and BCC samples in the vortex group were above 9, while RNA obtained from WB showed worse quality compared with BCC in the 4°C group. For verification using human blood, RIN values of frozen human WB rewarmed by vortexing ranged from 8.0 to 9.1. Blood components and rewarming methods could affect the RNA quality of blood samples. For scenarios where WB samples have already been cryopreserved, the vortex rewarming method is optimal for high-quality RNA. Otherwise, we would recommend centrifuging fresh WB and cryopreserving it in the form of BCC, which showed a tendency to obtain high-quality RNA by either of the two rewarming methods.