2.11 PCR-based gene expression studies
Expression studies were performed via semi-quantitative PCR and real
time quantitative PCR (RT-qPCR) analyses. For semi-quantitative PCR,
specific primers (mentioned in Table S1) were used to amplify
the core cDNA fragments. The PCR cycles were followed as described in
Table S1. PCR amplified products were separated in 1.5% agarose gel.
Gel-images of PCR products were photographed and analysed by ImageJ
software to compare the expression levels among the samples using actin
as reference gene.
RT-qPCR analysis for amplification of specific end-product genes was
performed by the steps as follows: initial denaturation at 94°C for 5
min and then following a cyclic reaction of 40 cycles (denaturation at
94°C for 45 s and annealing at 55°C for 1 min) using Power
SYBR® Green PCR master mix (applied biosystems by life
technologies). The relative gene expression level was calculated from
the 2-ΔΔCt method (Schmittgen and Livak, 2008) using
actin as the reference gene (Yu et al., 2017), which is constantly
expressed in floral tissue of jasmine.