文章基本信息

标题：Technical Variations in Low-Input RNA-seq Methodologies
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作者：Vipul Bhargava ; Steven R. Head ; Phillip Ordoukhanian 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2014
卷号：4
DOI：10.1038/srep03678
出版社：Springer Nature
摘要：Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.