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The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

ABSTRACT: AIMS:To establish whether RNA degrades in long-term storage at -80°C and whether RNA integrity numbers (RINs) determine 'fitness for purpose' in severely degraded RNA. METHODS:RNA was extracted from 549 thyroid biospecimens stored at -80°C for 0.1-10.9?years then their RINs correlated with storage time. RT-PCR for 65, 265, 534 and 942 base pair amplicons of hydroxymethylbilane synthase was used to measure amplicon length in RNA from cryopreserved and FFPE biospecimens that were equally degraded according to RIN. RESULTS:Storage time did not correlate with RIN. Longer amplicons were obtained from cryopreserved samples than FFPE samples with equal RINs. CONCLUSIONS:RNA does not degrade in thyroid biospecimens stored for long periods of time at -80°C. Although RINs are known to predict amenability to analytical platforms in good quality samples, this prediction is unreliable in severely degraded samples.

SUBMITTER: Mathieson W

PROVIDER: S-EPMC5637094 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

Mathieson William W Betsou Fay F Myshunina Tamara T Pushkarev Viktor V Pushkarev Vladimir V Shinkarkina Anna A Voskoboynyk Laryssa L Thomas Gerry A GA

Journal of clinical pathology 20160527 12

<h4>Aims</h4>To establish whether RNA degrades in long-term storage at -80°C and whether RNA integrity numbers (RINs) determine 'fitness for purpose' in severely degraded RNA.<h4>Methods</h4>RNA was extracted from 549 thyroid biospecimens stored at -80°C for 0.1-10.9 years then their RINs correlated with storage time. RT-PCR for 65, 265, 534 and 942 base pair amplicons of hydroxymethylbilane synthase was used to measure amplicon length in RNA from cryopreserved and FFPE biospecimens that were eq ...[more]

PMID: 27235537

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Project description:BackgroundThe 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.MethodsMaternal 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.ResultsOverall, 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.ConclusionsRNA 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.

Dataset Information

The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

Publications

The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

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