Expression profiling by high throughput sequencing
Summary
Based on the different chemistries of phenol extraction of RNA versus commercial kits, we hypothesized that certain species of mRNA may be preferentially extracted depending upon method, which could masquerade as differential expression in downstream RNA-seq experiments. We tested this using Saccharomyces cerevisiae samples that only differ in the RNA isolation method: a "standard" hot phenol extraction, versus two different commercial RNA isolation kits. While the kits had comparable relative mRNA abundances, samples isolated with hot phenol had higher relative abundance of mRNAs encoding membrane proteins. We hypothesize that hot phenol better solubilizes mRNAs associated with cellular membranes. We then compared the effects of each RNA isolation method on the ability to identify differentially expressed transcripts, using the yeast heat shock response a test case. The method of RNA isolation had little effect on the ability to identify differentially expressed transcripts. Thus, experiments within a single lab are unlikely to be affected by the choice of RNA isolation method as long as the same method is used throughout an experiment. For meta-analyses however, researchers should be cautious if trying to compare experiments where the RNA isolation methods differ.
Overall design
We performed RNA-seq comparing the effects of RNA isolation method on relative transcript abundance and the ability to detect differential expression.
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