Real-time quantitative RT-PCR assay of gene expression in plant roots during fungal pathogenesis

Real-time quantitative RT-PCR is becoming the preferred method for high-sensitivity, rapid-throughput RNA transcript quantification. However, due to the significant developmental costs of dedicated fluorogenic probes, a real-time assay that is simple to establish, comparatively inexpensive, and readily adaptable would be advantageous for the detailed analysis of large sets of expressed sequences. We have devised a flexible real-time quantitative RT-PCR assay that employs a nonspecific DNA binding dye for product detection and uses a relative quantification formula to account for differences in PCR amplification efficiency between the target and reference products. The latter permits the use of an exogenous reference transcript and therefore avoids the normal requirement for the construction of a recombinant RNA reference transcript or extensive characterization of housekeeping gene expression. In an investigation of class II chitinase expression in two varieties of Bermuda grass (Cynodon spp.), following infection with the fungal root pathogen Ophiosphaerella narmari, this assay identified 16- and 28-fold peaks in gene expression at 24 and 96 h after inoculation, respectively.