Comparison of RNA isolation methods on RNA-Seq: implications for differential expression and meta-analyses

Abstract

Background: The increasing number of transcriptomic datasets has allowed for meta-analyses, which can be valuable due to their increased statistical power. However, meta-analyses can be confounded by so-called "batch effects," where technical variation across different batches of RNA-seq experiments can clearly produce spurious signals of differential expression and reduce our power to detect true differences. While batch effects can sometimes be accounted for, albeit with caveats, a better strategy is to understand their sources to better avoid them. In this study, we examined the effects of RNA isolation method as a possible source of batch effects in RNAseq design. Results: Based on the different chemistries of "classic" hot phenol extraction of RNA compared to common commercial RNA isolation kits, we hypothesized that specific mRNAs may be preferentially extracted depending upon method, which could masquerade as differential expression in downstream RNA-seq analyses. We tested this hypothesis using the Saccharomyces cerevisiae heat shock response as a well-validated environmental response. Comparing technical replicates that only differed in RNA isolation method, we found over one thousand transcripts that appeared "differentially" expressed when comparing hot phenol extraction with the two kits. Strikingly, transcripts with higher abundance in the phenol-extracted samples were enriched for membrane proteins, suggesting that indeed the chemistry of hot phenol extraction better solubilizes those species of mRNA. Conclusions: Within a self-contained experimental batch (e.g. control versus treatment), the method of RNA isolation had little effect on the ability to identify differentially expressed transcripts. However, we suggest that researchers performing meta-analyses across different experimental batches strongly consider the RNA isolation methods for each experiment.

Prime-seq, efficient and powerful bulk RNA-sequencing

“…Hence, bead-based extraction does create a different expression profile than column based extraction, especially due to the often necessary Proteinase K incubation step. This confirms the general influence of RNA extraction protocols on gene expression profiles [51]. Importantly, the complexity of the two types of libraries is similar, with a slightly higher number of genes detected 15.…”

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank

“…To explain such discrepancies, we suggest that the β-actin mRNA is more sensitive to degradation by RNases or has a shorter half-life in these tissues, as it was shown previously in human cell lines 29 . In addition, we cannot exclude that RNA isolation methods differentially influence the recovery of some mRNA species, as previously suggested in studies comparing phenol extraction or RNA isolation kits 30 , 31 . For adipose tissue, Cts remains unchanged regardless RIN observed.…”

Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

“…The three RNA isolation methods analyzed here showed that the hot acid phenol method has not been used so far in transcriptional studies of this species (Table S1), even though it is considered an efficient RNA extraction method that has been used in other organisms (Scholes and Lewis, 2020;De Souza et al, 2021). For the CTAB method, according to the search performed on the Web of Science and Scopus databases, this method also has not been used in transcriptional studies of sweet potato.…”