Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues

BMC Biotechnology

Research Article

Jonatan Halvardson¹  Lucia Cavelier¹  Ammar Zaghlool¹  Lars Feuk¹  Adam Ameur¹  Linnea Nyberg¹  Manfred Grabherr² 

[1] Department of Immunology, Genetics and Pathology, Rudbeck Laboratory and Science for Life Laboratory, Uppsala University, Uppsala, Sweden;Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden;

关键词: RNA sequencing;    Transcriptomics;    RNA splicing;    RNA purification;    PolyA+ selection;    Cytoplasmic RNA;    Nuclear RNA;    Nascent transcripts;    De novo assembly;    Transcription profiling;   

DOI  :  10.1186/1472-6750-13-99

 received in 2013-07-08, accepted in 2013-11-04,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsWe 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.ConclusionOur 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.

【 授权许可】

Unknown   
© Zaghlool et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.