Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues
2013 (English)In: BMC Biotechnology, ISSN 1472-6750, Vol. 13, 99- p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2013. Vol. 13, 99- p.
Cuyoplasmic RNA, Nuclear RNA, RNA sequencing, mRNA
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-209270DOI: 10.1186/1472-6750-13-99ISI: 000327430800001OAI: oai:DiVA.org:uu-209270DiVA: diva2:656557