Comparison of relative RT-PCR and northern blot analyses to measure expression of beta-1,3-glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum

摘要

Although northern blot analysis is effective for quantifying gene expression, reverse transcription-polymerase chain reaction (RT-PCR) is much more sensitive. Obtaining quantitative RT-PCR results, however, can be challenging. Relative RT-PCR uses standard PCR techniques but permits the comparison of transcript quantities between samples by coamplifying the gene of interest with a housekeeping gene that acts as an internal control. To analyze the expression of a plant gene encoding a pathogenesis-related protein, such as beta-1,3-glucanase, a translation elongation factor 1alpha (EF-1alpha) gene was selected as an internal control. Northern blot analysis demonstrated constitutive expression of the plant EF-1alpha gene following infection of Nicotiana benthamiana by Colletotrichum destructivum. Primers for the gene of interest and internal control were compatible, and 35 cycles of amplification gave reproducible relative RT-PCR results for beta-1,3-glucanase gene expression. A high correlation was observed between the relative quantification of beta-1,3-glucanase gene expression as determined by northern blot and relative RT-PCR analyses, demonstrating the validity of relative RT-PCR with a plant EF-1alpha gene as a control.