Sequence detection software sds v2

RT-PCR was done using the RT-PCR Master Mix (USB) following the manufacturer’s instructions. For cDNA synthesis Superscript III reverse transcriptase (Invitrogen) and random primers (oligo(dN)6) according to the manufactures instructions were used. The RNA concentration was approximately 100 ng/µl and 10 µl was used in a total volume of 20 µl. The qRT-PCR was performed on an ABI PRISM 7300 Sequence Detector and results were calculated using the Sequence Detection Software SDS v2.0 (Applied BioSystems). Each qRT-PCR sample contained 12.5 µl Platinum SYBR Green qPCR SuperMix with UDG and ROX (Invitrogen), 2 mM MgCl₂, 0.5 µl forward and reverse primer (10 µM), 2 µl cDNA and water to make a 25 µl total reaction volume. The primer pairs used for qRT-PCR for WRKY33 were WRKY33qRTfor and WRKY33qRTrev (Table S1). Dissociation runs were performed to exclude the formation of primer dimers. The 18S gene was used as an internal reference and relative expression was calculated by the (1+E)^−ΔΔCt method [44] (link).

Semi quantitative RT-PCR was done using the RT-PCR Master Mix (USB) following the manufacturer’s instructions. For cDNA synthesis Superscript III reverse transcriptase (Invitrogen) and random primers (dN₆), according to the manufacturer’s instructions, were used. The qRT-PCR was performed on an ABI PRISM 7300 Sequence Detector (Applied BioSystems, Thermos Fisher Scientific, Vienna, Austria). Each qRT-PCR sample contained 12.5 μL Platinum SYBR Green qPCR SuperMix (Thermos Fisher Scientific, Vienna, Austria), 2 mM MgCl₂, 0.5 μL forward and reverse primer (10 μM) each, 2 μL cDNA, and water to make a total reaction volume of 25 μL. The RT-PCR and qRT-PCR forward and reverse primer pairs used for different genes are given in Table S3. Primer efficiencies were determined by using different concentrations of cDNA from seedlings of wild type. Control reactions with no cDNA template ruled out false positives. Dissociation runs were performed to make sure that there was no formation of primer dimers. The 18S gene was used as an internal reference in all qRT-PCR reactions. The results were calculated using the Sequence Detection Software SDS v2.0 (Applied BioSystems, Thermos Fisher Scientific, Vienna, Austria). The relative expression was calculated by the (1+E)^−ΔΔCt method [47 (link)].