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

IntroductionBlood 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.MethodsBefore RNA purification, blood samples from volunteers were exposed to 4 °C for 2, 6, 12, 24, or 48 h, 3 days, or 1 week, or exposed to room temperature (22–30 °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 NanoDrop™ 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, HIF1α, 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).ResultsFor blood samples stored at 4 °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 °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, HIF1α, HMOX1, and MKI67 in whole blood stored under different conditions showed irregular changes, suggesting that preservation conditions are also important for gene expression.ConclusionRNA integrity was qualified for blood samples stored at 4 °C for up to 72 h or at room temperature (22–30 °C) for up to 2 h. Hemolysis usually does not affect the RNA quality of blood samples unless the hemolysis method damages leukocytes.