Quality Control of RNA Extracted from PAXgene® Blood RNA Tubes after Long-Term Cryopreservation

Abstract

Background:

To systematically evaluate the long-term stability of cryopreserved RNA, we extended RNA quality monitoring in our renal biobank from 7 to 11 years for samples stored in PAXgene® Blood RNA tubes at −80°C.

Materials and Methods:

We assessed the suitability of archived PAXgene® RNA tubes for RNA sequencing by performing quality control on 217 chronic kidney disease samples, stratified by storage duration 7 (n = 62), 9 (n = 98), and 11 (n = 57) years. RNA was extracted from 2.5 mL whole blood using the PAXgene® Blood RNA Kit, with quality assessed based on concentration (Qubit™ Fluorometer, yield), purity (NanoDrop™ 2000 spectrophotometer, A_260/A280 and A_260/230 ratios), and integrity (Agilent 2100 Bioanalyzer, RNA integrity number, RIN). Sequencing eligibility required ≥500 ng total RNA and RIN ≥6.0.

Results:

Median RNA yields were comparable across storage durations (7-year 7.00 µg, 9-year: 7.11 µg, and 11-year: 6.79 µg, p = 0.870). The median A_260/280 ratios were 2.03 (7-year), 2.08 (9-year), and 2.07 (11-year) (p < 0.001, all ≥1.8), while median A_260/230 ratios were 1.72, 1.77, and 1.87, respectively (p = 0.550). RNA integrity, as measured by RIN, showed median values of 8.90 (7-year), 9.00 (9-year), and 8.80 (11-year). While no significant differences were observed between the 7- and 9-year (p = 0.537) or 7- and 11-year groups (p = 0.052), the 9-year group had slightly higher RIN values than the 11-year group (p < 0.05). Sequencing suitability remained consistently high (7-year: 97%, 9-year: 98%, and 11-year: 98%, p = 0.750), with 98% (212/217) of samples meeting the standards. Even under stricter RIN thresholds, pass rates remained robust (RIN ≥ 7.0: 94%, RIN ≥ 8.0: 88%).

Conclusion:

PAXgene® Blood RNA tubes stored at −80°C for up to 11 years provide high-quality RNA suitable for total RNA sequencing.