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首页> 外文期刊>BMC Biotechnology >Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues
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Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues

机译：高效的细胞分级分离可改善人类组织中成熟和新生转录本的RNA测序分析

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Background The starting material for RNA sequencing ( RNA-seq ) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression. Results We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq , making it suitable for analysis of nascent transcripts and RNA processing dynamics. Conclusion Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.
机译：背景技术用于RNA测序（RNA-seq）研究的起始材料通常是总RNA或polyA + RNA。两种形式的RNA代表成熟和加工水平不同的RNA分子异质库。除了与polyA +纯化步骤相关的偏见外，这种异质性还可能影响RNA-seq数据的分析，灵敏度和解释。我们假设RNA的亚细胞部分可能会提供更准确的基因表达图。结果我们提供了组织样品的细胞分级分离后细胞质和核RNA测序的结果。与传统的polyA + RNA相比，胞质RNA包含显着更高的外显子序列比例，从而在表达分析和剪接连接检测以及改进的RNA-seq数据从头组装过程中提供更高的灵敏度。相反，与总RNA-seq相比，核级分显示未处理的RNA富集，使其适用于新生转录本和RNA处理动力学的分析。结论我们的结果表明，在研究成熟的RNA时，与传统的polyA +富集相比，细胞分级分离是一种更快速，更具成本效益的方法。因此，分离的胞质和核RNA的RNA-seq可以显着改善来自哺乳动物组织的复杂转录组的分析。

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