Comparison of RNA Isolation Methods on RNA-Seq: Implications for Differential Expression and Meta-Analyses

Abstract

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. Thus, it is important to identify major sources of these so-called \"batch effects\" to eliminate them from study design. Based on the different chemistries of \"classic\" 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 and found that phenol extraction preferentially isolated membrane-associated mRNAs, thus resulting in spurious signals of differential expression. 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.