Quantitative Analysis of GUS Expression in Transgenic Plants

The expression levels of GUS mRNA in transgenic tobacco and Arabidopsis plants developed for the plasmids pKCaMV35SGUS and pSiM24GUS were evaluated by real-time quantitative RT-PCR [38] (link) using GUS-specific forward (5′-d-TTACGTCCTGTAGAAACCCCA-3′) and reverse (5′-d-ACTGCCTGGCACAGCAAT TGC-3′) primers. The qPCR assays were performed using the iTaq SYBR Green Supermix with ROX (Bio-Rad, USA) according to the manufacturer's instructions. Tobacco tubulin (by using forward 5′-d-ATGAGAGAGTGCATATCGAT-3′ and reverse 5′-d-TTCACTGAAGAAGGTGTTGAA-3′ primers) was used as an internal control to normalize the expression of GUS. The comparative threshold cycle (Ct) method (Applied Biosystems bulletin, part No. 4376784 Rev. C, 04/2007) was used to evaluate the relative expression levels of the transcripts. The threshold cycle was automatically determined for each reaction by the system set with default parameters (Step One Real-Time PCR System, Applied Biosystems). The specificity of the PCR was determined by melting curve analysis of the amplified products using the standard method installed in the system (Step One Real-Time PCR System, Applied Biosystems).