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.