BMC Biotechnology | |
Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues | |
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 |
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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.
【 预 览 】
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20140715100852770.pdf | 717KB | download | |
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Figure 3. | 42KB | Image | download |
Figure 2. | 75KB | Image | download |
Figure 1. | 80KB | Image | download |
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