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Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome
Year:
2009
Source of publication :
Nature Protocols
Authors :
German, Marcelo A.
;
.
Volume :
4
Co-Authors:
German, M.A., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Luo, S., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Schroth, G., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Meyers, B.C., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Green, P.J., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Facilitators :
From page:
356
To page:
362
(
Total pages:
7
)
Abstract:
We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5’-rapid amplification of cDNA ends, deep sequencing of 3’ cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5’-RNA adapter that includes an MmeI recognition site is ligated to 5’- monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5’ equally-sized fragments are gel-selected, ligated to a 3' doublestranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6-7 d. © 2009 Macmillan Publishers Limited. All rights reserved.
Note:
Related Files :
Animal
Animals
article
biology
Computational Biology
Genetics
methodology
microRNA
sequence analysis
Sequence Analysis, RNA
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Related Content
More details
DOI :
10.1038/nprot.2009.8
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
29648
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:48
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Scientific Publication
Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome
4
German, M.A., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Luo, S., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Schroth, G., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Meyers, B.C., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Green, P.J., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome
We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5’-rapid amplification of cDNA ends, deep sequencing of 3’ cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5’-RNA adapter that includes an MmeI recognition site is ligated to 5’- monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5’ equally-sized fragments are gel-selected, ligated to a 3' doublestranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6-7 d. © 2009 Macmillan Publishers Limited. All rights reserved.
Scientific Publication
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