Gene expression analysis by real-time quantitative PCR

Total RNA was extracted from the plant tissues grown in the control or various stress conditions using an RNeasy kit (Qiagen, Amsterdam, Netherlands). One μg of RNA from each sample was used in the reverse transcription with a cDNA synthesis kit (Code No.: AH311-02, TransGen, Beijing, China) according to the instructions provided by the manufacturer. Real-time quantitative PCR (qPCR) was conducted on an ABI 7500 system (Applied Biosystems, New York, USA) under the default thermal cycling conditions using a TransStart^TM Green qPCR SuperMix kit (TransGen) and specific primers for PCR amplification. Relative transcript abundance was determined and normalized with the reference gene Actin2 (GenBank Accession No. {"type":"entrez-nucleotide","attrs":{"text":"U41998","term_id":"1669386","term_text":"U41998"}}U41998) to minimize variation in the levels of the cDNA templates. For expression profile examination of TsApx6, AtApx6 and stress-related genes under salt stress conditions, the control sample acted as the calibrator with a nominal value of 1. When investigated the Gus expression, Actin2 itself was calibrated with a nominal value of 100. The data represent mean values derived from at least three independent repetitive amplifications and each experiment was conducted in at least three biological replicates. All calculations and analyses were performed by the 2^-△△Ct method. The primers used in qPCR of TsApx6, AtApx6, Actin2 and the stress/ABA-responsive genes are shown in Table A in S1 Text.