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Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.
Institute of Medical Science, University of Tokyo, Japan.
We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).
PMID: 8125298 [PubMed - indexed for MEDLINE]
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Cited by 71 PubMed Central articles
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High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.
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PLoS One. 2009; 4(1):e4108. Epub 2009 Jan 1.
[PLoS One. 2009]
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Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library.
Umezawa T, Sakurai T, Totoki Y, Toyoda A, Seki M, Ishiwata A, Akiyama K, Kurotani A, Yoshida T, Mochida K, et al.
DNA Res. 2008 Dec; 15(6):333-46. Epub 2008 Oct 16.
[DNA Res. 2008]
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MachiBase: a Drosophila melanogaster 5'-end mRNA transcription database.
Ahsan B, Saito TL, Hashimoto S, Muramatsu K, Tsuda M, Sasaki A, Matsushima K, Aigaki T, Morishita S.
Nucleic Acids Res. 2009 Jan; 37(Database issue):D49-53. Epub 2008 Oct 8.
[Nucleic Acids Res. 2009]
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