4.4. RNA Purification and Expression Analyses with Quantitative Real-Time PCR

The expression rate of Arabidopsis GST genes was determined by quantitative real-time PCR (RT-qPCR) after the purification of RNA from 100 mg plant material according to Chomczynski and Sacchi [57] as was described in Bela et al. [40]. The primers used for the RT-qPCR are given in Table S3. The expression rate of GST genes was monitored as published earlier in Bela et al. [40]. Data analysis was performed using qTOWER Software 2.2 (Analytik Jena, Jena, Germany) software. The GAPDH2 (At1g13440) and actin2 (At3g18780) genes were used as internal controls, respectively [17]. The average Ct number of GAPDH2 and actin2 gene was used for data normalization. Data of RT-qPCR was calculated using the 2^(-∆∆Ct) formula [58]. To demonstrate the differences between changes in the expression levels of the investigated GST genes, the relative transcript level in the Arabidopsis thaliana Col-0 control root samples were considered to be one for each gene, and the results were presented on a heat map created in Microsoft Excel using the conditional formatting function [40]. To demonstrate the differences in the enhancement of gene expression levels a lighter green colour indicates if the increase was between 2 - 2.99 and dark green if more than 3 fold elevation occurred compared to the level in the wild-type control.