Quality Assessment of RNA in Long-Term Storage: the All Our Families Biorepository.

Background The All Our Families (AOF) cohort study is a longitudinal population-based study which collected biological samples from 1948 pregnant women between May 2008 and December 2010. As the quality of samples can decline over time, the objective of the current study was to assess the association between storage time and RNA (ribonucleic acid) yield and purity, and confirm the quality of these samples after 7–10 years in long-term storage. Methods Maternal whole blood samples were previously collected by trained phlebotomists and stored in four separate PAXgene Blood RNA Tubes (PreAnalytiX) between 2008 and 2011. RNA was isolated in 2011 and 2018 using PAXgene Blood RNA Kits (PreAnalytiX) as per the manufacturer’s instruction. RNA purity (260/280), as well as RNA yield, were measured using a Nanodrop. The RNA integrity number (RIN) was also assessed from 5–25 and 111–130 months of storage using RNA 6000 Nano Kit and Agilent 2100 BioAnalyzer. Descriptive statistics, paired t-test, and response feature analysis using linear regression were used to assess the association between various predictor variables and quality of the RNA isolated. Results Overall, RNA purity and yield of the samples did not decline over time. RNA purity of samples isolated in 2011 (2.08, 95% CI: 2.08–2.09) were statistically lower (p<0.000) than samples isolated in 2018 (2.101, 95% CI: 2.097, 2.104), and there was no statistical difference between the 2011 (13.08 μg /tube, 95% CI: 12.27–13.89) and 2018 (12.64 μg /tube, 95% CI: 11.83–13.46) RNA yield (p = 0.2964). For every month of storage, the change in RNA purity is -0.01(260/280), and the change in RNA yield between 2011 and 2018 is -0.90 μ g / tube. The mean RIN was 8.49 (95% CI:8.44–8.54), and it ranged from 7.2 to 9.5. The rate of change in expected RIN per month of storage is 0.003 (95% CI 0.002–0.004), so while statistically significant, these results are not relevant. Conclusions RNA quality does not decrease over time, and the methods used to collect and store samples, within a population-based study are robust to inherent operational factors which may degrade sample quality over time.