长期储存 RNA 的质量评估：All Our Families 生物库。
Quality assessment of RNA in long-term storage: The All Our Families biorepository.
机构信息
Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
出版信息
PLoS One. 2020 Dec 1;15(12):e0242404. doi: 10.1371/journal.pone.0242404. eCollection 2020.
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.
背景
All Our Families（AOF）队列研究是一项基于人群的纵向研究，该研究于 2008 年 5 月至 2010 年 12 月期间从 1948 名孕妇中采集了生物样本。由于样本的质量随时间推移而下降，因此本研究的目的是评估储存时间与 RNA（核糖核酸）产量和纯度之间的关系，并确认这些样本在长期储存 7-10 年后的质量。
方法
研究人员此前通过训练有素的采血员采集了孕妇的全血样本，并将其储存在四个单独的 PAXgene Blood RNA Tubes（PreAnalytiX）中，时间范围为 2008 年至 2011 年。2011 年和 2018 年按照制造商的说明使用 PAXgene Blood RNA Kits（PreAnalytiX）分离 RNA。使用 Nanodrop 测量 RNA 纯度（260/280）以及 RNA 产量。还使用 RNA 6000 Nano Kit 和 Agilent 2100 BioAnalyzer 评估了 5-25 个月和 111-130 个月储存时的 RNA 完整性号（RIN）。使用描述性统计、配对 t 检验和线性回归响应特征分析来评估各种预测变量与分离的 RNA 质量之间的关系。
结果
总体而言，样本的 RNA 纯度和产量随时间推移而没有下降。2011 年分离的样本的 RNA 纯度（2.08，95%置信区间：2.08-2.09）统计上低于 2018 年分离的样本（2.101，95%置信区间：2.097，2.104）（p<0.000），并且 2011 年（13.08μg/管，95%置信区间：12.27-13.89）和 2018 年（12.64μg/管，95%置信区间：11.83-13.46）的 RNA 产量没有统计学差异（p=0.2964）。每储存一个月，RNA 纯度的变化为-0.01（260/280），2011 年至 2018 年之间的 RNA 产量变化为-0.90μg/管。平均 RIN 为 8.49（95%置信区间：8.44-8.54），范围为 7.2 至 9.5。每个月储存时预期 RIN 的变化率为 0.003（95%置信区间 0.002-0.004），虽然具有统计学意义，但这些结果并不相关。
结论
RNA 质量不会随时间下降，并且在基于人群的研究中用于收集和储存样本的方法对于随时间推移可能降低样本质量的固有操作因素具有稳健性。
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