RNAlater and flash freezing storage methods nonrandomly influence observed gene expression in RNAseq experiments

Abstract

32RNA-sequencing is a popular next-generation sequencing technique for assaying 33 genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are 34 introduced by sample handling prior gene expression measurements. Two of the most 35 common methods for preserving samples in both field-based and laboratory conditions 36 are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid 37 nitrogen can be impractical, particularly for field collections. RNAlater is a solution for 38 stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular 39RNA. In this study, we assessed genome-wide expression patterns in 30 day old fry 40 collected from the same brood at the same time point that were flash-frozen in liquid 41nitrogen and stored at -80°C or submerged and stored in RNAlater at room 42 temperature, simulating conditions of fieldwork. We show that sample storage is a 43 significant factor influencing observed differential gene expression. In particular, genes 44 with elevated GC content exhibit higher observed expression levels in liquid nitrogen 45 flash-freezing relative to RNAlater-storage. Further, genes with higher expression in 46RNAlater relative to liquid nitrogen experience disproportionate enrichment for 47 functional categories, many of which are involved in RNA processing. This suggests 48that RNAlater may elicit a physiological response that has the potential to bias biological 49interpretations of expression studies. The biases introduced to observed gene 50 expression arising from mimicking many field-based studies are substantial and should 51 not be ignored. 52 53

Comparative Evaluation of RNAlater Solution and Snap Frozen Methods for Gene Expression Studies in Different Tissues

“…Although RNAlater contributes to the prevention of RNA degradation by limiting the RNase activity, its effect on gene activity is not exactly known when the tissues are stored in RNAlater at +4°C. A study has reported that when tissues are stored in the RNAlater, there were increases or decreases in the activity of about 3,000 genes (15). This may be caused by the fact that gene activity is not inhibited during fixation of tissues with RNAlater.…”

“…There are studies using standard methods (electrophoretic and spectrophotometric) to control the quality of RNA in tissues stored with RNAlater (15)ite></EndNote>. Although there are some studies comparing the quantity of genes through Ct values by means of RT-qPCR in addition to standard controls, studies performing their examinations at the level of gene expression are limited (15,16). In this study, possible RNA quality loss that may occur in the lung, heart, liver, and skeletal muscle tissues stored in RNAlater was studied in terms of Ct and gene expression through electrophoretic and spectrophotometric controls as well as PPIA (Peptidylprolyl Isomerase A), ACTB (Beta Actin), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), and SRP72 (Signal Recognition Particle 72) genes, which are commonly used as reference genes (17)(18)(19)(20)(21).…”

Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

“…Others account for "unwanted variance" due to technical, batch, or experimental variation. In contrast, the in uence that sample handling requirements , such as tissue lysis or varying processing times [12][13][14] , have on RNA-seq measurement quality is not comprehensively characterized. Knowledge-gaps in sample-handling impact can make it di cult to control for such factors.…”

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

“…The majority of short-read RNA-Seq studies document host responses to different perturbations or compare different cell types and tissues, each approach requiring a careful consideration of the experimental design to avoid batch effects and other confounding influences that are discussed in detail elsewhere (16,17). Standard short-read RNA-Seq pipelines generate tens of millions of paired-end reads that are then aligned to the host genome and/or transcriptome.…”