Abstract
ObjectiveLong non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at − 80 °C. Here we sought to determine the effects on lncRNA expression profiles following − 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze–thaw cycles on both total RNA and cDNA obtained from PAXgene tubes.ResultsWe find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at − 80 °C or up to ten total RNA or cDNA freeze–thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.
Highlights
Long non-coding RNAs play pivotal roles in gene regulation, protein synthesis, sex chromosome compensation and telomere maintenance [1–5]
We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at − 80 °C or up to ten total RNA or cDNA freeze–thaw cycles do not significantly alter Long non-coding RNA (lncRNA) expression profiles compared to baseline
Evaluation of lncRNA expression following storage of PAXgene tubes, RNA and cDNA for up to one year To examine the effects of various storage conditions on lncRNA expression, we initiated our study utilizing a panel of annotated lncRNAs from previous RNA sequencing studies that were conducted across multiple human autoimmune diseases (Additional file 1: Table S1 and Additional file 2: Fig.S1)
Summary
Evaluation of lncRNA expression following storage of PAXgene tubes, RNA and cDNA for up to one year To examine the effects of various storage conditions on lncRNA expression, we initiated our study utilizing a panel of annotated lncRNAs from previous RNA sequencing studies that were conducted across multiple human autoimmune diseases (Additional file 1: Table S1 and Additional file 2: Fig.S1). During the course of the study, consecutive, monthly assessments were performed whereby five PAXgene tubes were pulled from storage and expression levels of each lncRNA was analyzed for a total of twelve series of qRT-PCR measurements (Additional file 4: Fig. S3). Evaluation of RNA integrity number, lncRNA and mRNA expression following freeze–thaw cycles We investigated the effects of long-term storage on RNA integrity and the impact of multiple freeze–thaw cycles on total RNA by analyzing RNA integrity number (RIN) values as depicted in Additional file 7: Fig.S5 These values are calculated comparing 18S and 28S rRNA ratios. Canonical protein-coding genes were measured to test whether − 80 °C storage or up to 10 freeze–thaw cycles would significantly alter expression of these mRNA targets as well (Fig. 3b) These mRNAs were selected from the same study comparing healthy control subjects and patients with relapsing–remitting multiple sclerosis [16, 18]. Similar results were observed when cDNA samples were exposed to five or ten freeze–thaw cycles (Additional file 10: Fig.S8)
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