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Anal Biochem. 2015 Sep 1;484:72-4. doi: 10.1016/j.ab.2015.04.039. Epub 2015 May 8.

CAP(+) selection: A combined chemical-enzymatic strategy for efficient eukaryotic messenger RNA enrichment via the 5' cap.

Author information

1
Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CH-1211 Geneva, Switzerland.
2
Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CH-1211 Geneva, Switzerland; Institute of Genetics and Genomics in Geneva (iGE3), University of Geneva, CH-1211 Geneva, Switzerland. Electronic address: joseph.curran@unige.ch.

Abstract

Eukaryotic messenger RNAs (mRNAs) are generally enriched using oligo(dT) selection. However, a significant fraction of mRNAs contain either short or no poly(A). Our technique permits the isolation of mRNAs via their unique biochemical feature, the 5' cap. It involves RNA extraction, blocking of the 3' ribose cis-diol by cordycepin, oxidation of the 5' cis-diol of the CAP to a dialdehyde, coupling to a biotinylated linker, and enrichment on a streptavidin affinity matrix. We demonstrate that it efficiently pulls out a synthetic capped and non-polyadenylated transcript used to spike total cell RNA as well as endogenous histone 3c mRNA reported to be poly(A) negative.

KEYWORDS:

5′ Cap; Biotinylation; Purification; mRNA

PMID:
25963893
DOI:
10.1016/j.ab.2015.04.039
[Indexed for MEDLINE]

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