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 RNA-seq design. 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. 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.

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RNA-Seq上RNA分离方法的比较：差异表达和荟萃分析的意义

转录组数据集数量的增加允许进行荟萃分析，由于其统计能力的提高，这可能是有价值的。但是，荟萃分析可能会与所谓的“批处理效应”混淆，在批处理效应中，不同批次的RNA-seq实验之间的技术差异会明显产生差异表达的虚假信号，并降低我们检测真实差异的能力。尽管有时需要注意一些事项，但有时需要考虑批次效应，但更好的策略是了解其来源，以更好地避免它们。在这项研究中，我们检查了RNA分离方法的效果，认为它是RNA-seq设计中批量效应的可能来源。基于与常规的商用RNA分离试剂盒相比，“经典”热酚提取RNA的不同化学方法，我们假设根据方法可以优先提取特定的mRNA，这可能会伪装成下游RNA序列分析中的差异表达。我们使用酿酒酵母的热休克反应作为经过充分验证的环境反应来检验此假设。比较仅在RNA分离方法上有所不同的技术复制品，我们发现将热酚提取物与两种试剂盒进行比较时，有超过一千个转录物以“差异”表达。令人惊讶的是，在苯酚提取的样品中，具有较高丰度的转录物富含膜蛋白，这表明热酚提取的化学确实确实能更好地溶解那些mRNA。在一个独立的实验批次中（例如对照与治疗），RNA分离方法对鉴定差异表达转录本的能力影响很小。但是，我们建议研究人员在不同实验批次之间进行荟萃分析时，应强烈考虑每个实验的RNA分离方法。