Technical Variations in Low-Input RNA-seq Methodologies.

TitleTechnical Variations in Low-Input RNA-seq Methodologies.
Publication TypeJournal Article
Year of Publication2014
AuthorsBhargava V, Head SR, Ordoukhanian P, Mercola M, Subramaniam S
JournalSci Rep
Volume4
Pagination3678
Date Published2014
ISSN2045-2322
Abstract

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.

DOI10.1038/srep03678
PubMed URLhttp://www.ncbi.nlm.nih.gov/pubmed/24419370?dopt=Abstract
PMCPMC3890974
Alternate JournalSci Rep
PubMed ID24419370