Abstract
Real-time gene expression analysis by semi-quantitative and quantitative RT-PCR requires a set of gene-specific primers which should have the ability to amplify the gene of interest specifically. In the present study, we have standardized certain parameters for primer design using the freely available Primer3 software. We have designed the primers for defense genes such as ICS (isochorismate synthase), CCoAOMT (caffeoyl CoA O-methyltransferase), C4H (cinnamate 4-hydroxylase), and G-alpha in pea. We have also discussed, the way of sequence retrieval, when the sequence is not reported in the organism of interest. We have evaluated the designed primers using cDNA prepared from mRNA isolated from the pea leaves. By analyzing the results, we have found that primers are perfectly binding with the target and giving single sharp band on a DNA electrophoresis gel. It can be concluded that the parameters used for primer designing by Primer3 play a critical role in the experimental results and parameters defined in the present study resulting in a very good amplification during PCR.