Multiplex titration RT-PCR: rapid determination of gene expression patterns for a large number of genes.

摘要

An improved method for determination of gene expression levels using RT-PCR in plants is described. The procedure is rapid and does not require extensive optimization or densitometric analysis. The method includes a control primer pair in every reaction, and is robust enough to allow the inclusion of several primer pairs per reaction, thus markedly increasing efficiency. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, nine members of the Aux/IAA family of auxin-responsive genes from tomato (Lycopersicon esculentum) were rapidly screened, and genes which vary in message abundance in a tissue- and light-specific manner identified. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, this method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.