A multiplex RT-PCR assay for analysis of relative transcript levels of different members of multigene families: Application to Arabidopsis calmodulin gene family

Abstract

The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we describe a simple, semi-quantitative and single tube multiplex RT-PCR assay for simultaneous and relative expression analysis with an application to all the six members of Arabidopsis calmodulin multigene family. In the multiplex primer set, individual gene specific primers were derived from 3'-untranslated region of the genes and a single common primer from the conserved exonic region. Transcriptional activation of all the members of the calmodulin gene family in response to touch was monitored. The results demonstrate that two of the genes are not regulated by touch; however, the other four that are induced by touch show a differential response including their kinetics of induction.