Issue 5, 2017

Highly sensitive and multiplexed quantification of mRNA splice variants by the direct ligation of DNA probes at the exon junction and universal PCR amplification

Abstract

Alternative messenger RNA (mRNA) splicing is a basic mechanism of gene regulation. In general, reverse transcription and polymerase based primer extension limit the sensitivity and selectivity of the current detection of mRNA splice variants, respectively. Here, we show that, using the ligation of two properly designed probes at the exon junction combined with universal PCR amplification, as little as a single copy of a mRNA splice variant per cell can be accurately determined, and the dynamic range covers six orders of magnitude. Three mRNA splice variants were measured from total RNA samples derived from different cell lines. Moreover, by encoding the ligation probes with different lengths, multiplexed mRNA splice variants can be simultaneously detected in one-tube PCR amplification using electrophoretic separation.

Graphical abstract: Highly sensitive and multiplexed quantification of mRNA splice variants by the direct ligation of DNA probes at the exon junction and universal PCR amplification

Supplementary files

Article information

Article type
Edge Article
Submitted
09 Jan 2017
Accepted
01 Mar 2017
First published
01 Mar 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2017,8, 3635-3640

Highly sensitive and multiplexed quantification of mRNA splice variants by the direct ligation of DNA probes at the exon junction and universal PCR amplification

H. Wang, H. Wang, X. Duan, Y. Sun, X. Wang and Z. Li, Chem. Sci., 2017, 8, 3635 DOI: 10.1039/C7SC00094D

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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