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

BMC Biotechnology

Ammar Zaghlool²  Adam Ameur²  Linnea Nyberg²  Jonatan Halvardson²  Manfred Grabherr¹  Lucia Cavelier²  Lars Feuk² 

[1] Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

[2] Department of Immunology, Genetics and Pathology, Rudbeck Laboratory and Science for Life Laboratory, Uppsala University, Uppsala, Sweden

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

Others  :  835101
DOI  :  10.1186/1472-6750-13-99

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

【 摘 要 】

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.

【 授权许可】

   
2013 Zaghlool et al.; licensee BioMed Central Ltd.

【 图 表 】

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