Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

Abstract

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at −80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.

Comparative transcriptome analysis of wild and lab populations of Astyanax mexicanus uncovers differential effects of environment and morphotype on gene expression

“…Liver tissues from corresponding lab-raised populations were collected and preserved in RNAlater as fixative using the same methods as for wild-caught fish (Passow et al, 2019). The H&E staining of the liver sections showed normal hepatic architecture and no steatosis, reminiscent of healthy liver tissue ( Figure S3).…”

Sample Preservation and Storage Significantly Impact Taxonomic and Functional Profiles in Metaproteomics Studies of the Human Gut Microbiome

“…This study investigated the influence of two popular sample storage methods on protein identification rates in a mass spectrometry-based metaproteomics study of human faecal samples and subsequent information gain. Flash freezing of samples and subsequent storage at −80 °C is proven to be the storage method that keeps samples close to their original state after specimen collection [34,35]. Furthermore, RNAlater is a popular RNA stabilizing agent [36,37,38,39,40] that is also often used when other biomolecules in addition to RNA are extracted from the same sample [41,42].…”

Unique transcriptional signatures of sleep loss across independently evolved cavefish populations

“…Protocols for RNA extraction, library preparation, and sequencing were performed as previously described (Passow et al, 2019). To isolate RNA, whole 30‐dpf fry (~30 mg of tissue) were homogenized and then lysed.…”

“…Protocols for RNA extraction, library preparation, and sequencing were performed as previously described (Passow et al, 2019 (Table S1).…”