Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment

Introduction: Blood samples are the most common biospecimen in biobanks, and RNA from such blood samples is an important material for biomedical research. High-quality RNA is essential for consistent, reliable results. Preanalytical environmental conditions can affect the quality of blood RNA. Here, we carried out a quantitative assessment of the influence of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens in biobanks. Methods: Before RNA purification, blood samples from volunteers were exposed to 4 & DEG;C for 2, 6, 12, 24, or 48 h, 3 days, or 1 week, or exposed to room temperature (22-30 & DEG;C) for 1, 2, 6, 12, or 24 h. Hemolyzed samples were collected from laboratory department and some of them were prepared using the freeze-thaw method. After exposure to different preanalytical environmental conditions, the RNA simple Total RNA Kit was used to purify the RNA, following which a NanoDropTM One and Qsep 100 Bio-Fragment Analyzer were used to assess RNA concentration, purity, and integrity. In addition, a part of the RNA was immediately reverse transcribed into cDNA when it was purified, then the relative expression levels of 18S, ACTB, HIF1 & alpha;, HMOX1, and MKI67 were determined by real-time quantitative PCR. Finally, 30 blood samples were collected from the surplus samples in our laboratory department to assess their RNA quality without knowledge of their storage conditions (duration/temperature). Results: For blood samples stored at 4 & DEG;C, there was a significant difference between the RNA integrity after 1 week compared to after 2 h. For blood samples stored at room temperature (22-30 & DEG;C), the RNA integrity was also significantly different at 6 h and 0 h. Hemolysis caused by freeze-thawing severely affected RNA quality, whereas clinical hemolysis generally produced no significant effects. Moreover, expression of 18S, ACTB, HIF1 & alpha;, HMOX1, and MKI67 in whole blood stored under different conditions showed irregular changes, suggesting that preservation condi-tions are also important for gene expression. Conclusion: RNA integrity was qualified for blood samples stored at 4 & DEG;C for up to 72 h or at room temperature (22-30 & DEG;C) for up to 2 h. Hemolysis usually does not affect the RNA quality of blood samples unless the hemolysis method damages leukocytes.