期刊
METHODS
卷 118, 期 -, 页码 93-100出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2016.10.005
关键词
RNA-binding protein; RNA-protein interaction; Ribonucleoprotein complex; Post-transcriptional regulation; Antisense oligonucleotides; Non-coding RNA
资金
- Biotechnology and Biological Sciences Research Council [BB/K009303/1]
- Swiss National Science Foundation [CSRII3-141942]
- BBSRC [BB/K009303/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/K009303/1] Funding Source: researchfish
We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues. (C) 2016 Elsevier Inc. All rights reserved.
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