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

Zaghlool A, Ameur A, Nyberg L, Halvardson J, Grabherr M, Cavelier L, Feuk L

BMC Biotechnol. 13 (-) 99 [2013-11-13; online 2013-11-13]

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. 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. 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.

NGI Uppsala (Uppsala Genome Center)

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PubMed 24225116

DOI 10.1186/1472-6750-13-99

Crossref 10.1186/1472-6750-13-99

1472-6750-13-99

pmc PMC3833653