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 sequenci...
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| Published in | Nature protocols Vol. 4; no. 3; pp. 356 - 362 |
|---|---|
| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
01.02.2009
Nature Publishing Group |
| Subjects | |
| Online Access | Get full text |
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| 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
Mme
I 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
Mme
I. The 5′ equally-sized fragments are gel-selected, ligated to a 3′ double-stranded 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. |
|---|---|
| AbstractList | 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' double-stranded 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. 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 5' double-stranded 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. 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 Mme I 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 Mme I. The 5′ equally-sized fragments are gel-selected, ligated to a 3′ double-stranded 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. |
| Audience | Academic |
| Author | Luo, Shujun Meyers, Blake C Green, Pamela J German, Marcelo A Schroth, Gary |
| Author_xml | – sequence: 1 givenname: Marcelo A surname: German fullname: German, Marcelo A organization: Delaware Biotechnology Institute, University of Delaware – sequence: 2 givenname: Pamela J surname: Green fullname: Green, Pamela J organization: Delaware Biotechnology Institute, University of Delaware – sequence: 3 givenname: Shujun surname: Luo fullname: Luo, Shujun organization: Illumina Inc., 25861 Industrial Boulevard – sequence: 4 givenname: Gary surname: Schroth fullname: Schroth, Gary organization: Illumina Inc., 25861 Industrial Boulevard – sequence: 5 givenname: Blake C surname: Meyers fullname: Meyers, Blake C organization: Delaware Biotechnology Institute, University of Delaware |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/19247285$$D View this record in MEDLINE/PubMed https://www.osti.gov/biblio/973509$$D View this record in Osti.gov |
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| Snippet | We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse... |
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| SubjectTerms | Analytical Chemistry Animals BASIC BIOLOGICAL SCIENCES Bioinformatics Biological Techniques Biomedical and Life Sciences Computational Biology - methods Computational Biology/Bioinformatics Deoxyribonucleic acid DNA Filters Genetic research Life Sciences Methods Microarrays MicroRNAs MicroRNAs - genetics Organic Chemistry Physiological aspects Polymerase chain reaction protocol RNA RNA Stability Sequence Analysis, RNA - methods |
| Title | Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome |
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