Comparison of RNA isolation methods on RNA-Seq: implications for differential expression and meta-analyses
Comparison of RNA isolation methods on RNA-Seq: implications for differential expression and meta-analyses
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DOI：
10.1186/s12864-020-6673-2
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发表时间：
2020-03-20
期刊：
影响因子：
4.4
通讯作者：
Lewis, Jeffrey A.
中科院分区：
文献类型：
--
作者：
Scholes, Amanda N.;Lewis, Jeffrey A.
背景转录组数据集数量的增加使得荟萃分析成为可能，由于其统计能力的增强，荟萃分析非常有价值。然而，荟萃分析可能会受到所谓的“批次效应”的影响，即不同批次的 RNA-seq 实验之间的技术差异可能会明显产生差异表达的虚假信号，并降低我们检测真正差异的能力。虽然有时可以考虑批次效应（尽管有一些警告），但更好的策略是了解其来源以更好地避免它们。在本研究中，我们检查了 RNA 分离方法的影响，作为 RNA-seq 设计中批次效应的可能来源。结果基于与常见的商业 RNA 分离试剂盒相比，“经典”热苯酚提取 RNA 的不同化学物质，我们假设根据方法可能会优先提取特定的 mRNA，这些 mRNA 可能在下游 RNA-seq 分析中伪装成差异表达。我们使用酿酒酵母热休克反应作为经过充分验证的环境反应来测试这一假设。比较仅在 RNA 分离方法上不同的技术重复，我们发现在将热苯酚提取与两个试剂盒进行比较时，有超过 1000 个转录本出现“差异”表达。引人注目的是，苯酚提取样品中丰度较高的转录本富集了膜蛋白，这表明热苯酚提取的化学作用确实可以更好地溶解这些 mRNA。结论在独立的实验批次（例如对照与处理）中，RNA 分离方法对识别差异表达转录本的能力几乎没有影响。然而，我们建议研究人员在不同实验批次之间进行荟萃分析时，强烈考虑每个实验的 RNA 分离方法。
BackgroundThe 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.ResultsBased 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.ConclusionsWithin 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.