The Effect of Blood Clots on the Quality of RNA Extracted from PAXgene Blood RNA Tubes.

Abstract

Background: PAXgene^® Blood RNA tubes are routinely used in clinical research and molecular biology applications to preserve the stability of RNA in whole blood. However, in practice, blood clots are occasionally observed after blood collection and are often ignored. Currently, there are few studies on whether blood clots affect the quality of RNA extracted from these tubes. Materials and Methods: Fifteen pairs of non-clot and clot PAXgene Blood RNA tube samples (n = 30) were collected to form two matched groups from 15 patients. According to the maximum diameter (d) of the blood clot observed visually at the time of sample reception, the clot groups were divided into a small-clot group (0 cm < d < 0.5 cm) and a large-clot group (d ≥ 0.5 cm). RNA was extracted by the PAXgene Blood RNA Kit. To analyze the quality of RNA, its yield and purity were assessed by spectrophotometry, and integrity was measured by microfluidic electrophoresis. An A_260/280 ratio between 1.8 and 2.2 indicated purified RNA, and RNA integrity number (RIN) values ≥7.0 were considered to represent qualified integrity. Results: The median yields of RNA from the non-clot and clot groups were 3.84 (2.80-6.38) μg and 4.87 (2.77-8.30) μg, respectively. The median A_260/280 ratios were 2.08 (2.06-2.09) and 2.09 (2.07-2.11), whereas the median A_260/230 ratios were 1.77 (1.31-1.91) and 1.67 (1.21-1.94) in the two groups. In addition, the median RINs were 8.20 (8.00-8.40) and 7.20 (6.60-7.70), respectively. There were no significant differences in RNA yields, A_260/280, or A_260/230 between the two groups. However, the RIN value of the clot group was significantly lower compared with the non-clot group (p < 0.05), with RIN ≥7.0 found in all non-clot samples and 60% of clot samples (p < 0.05). Furthermore, in the clot groups, the small-clot samples had higher RIN values than large-clot samples (8.25 [7.75-8.75] vs. 6.90 [6.60-7.30], p < 0.001). Conclusions: The formation of large blood clots in PAXgene Blood RNA tubes will reduce the integrity of extracted RNA.