Sybr green pcr master mix system

Total RNA for each sample was isolated using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, Beijing, China) according to the manufacturer’s protocol. Total RNA was reversed to cDNA using an M-MLV Reverse Transcriptase Kit (Bioteke Corporation, Beijing, China). Quantitative Real-time PCR (qRT-PCR) was carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) in a 20 μL volume containing 100 ng of cDNA, 4 pM of each primer, and 10 μL SYBR Green PCR Master Mix system (TaKaRa). The PCR conditions and the calculation method of gene expression were the same as what had been described previously [68 (link)]. Information on the qRT-PCR primers for gene expression analysis and gene cloning used in this study was listed in S3 Table. The nucleotide sequences of GhMFT homoeologous genes marked with primer location for qRT-PCR were shown in S1 Fig. A cotton Ubiquitin7 (GhUBQ7, GenBank accession no. DQ116441) gene and an Arabidopsis Actin2 (AT3G18780) gene were used as internal controls, respectively. Three replicate assays were conducted with separately isolated RNA, and three technical triplicates were performed for each PCR reaction.

Three biological replications with two technique replications of RNA were used for qPCR analysis. After being treated with RNase-free DNase, RNA samples were used as templates for reverse transcription with the M-MLV RTase cDNA Synthesis Kit (Takara, Dalian, China). Primers were designed using PRIMER3 software and listed in S1 Table. The expression of β-actin gene was used as a control. About 1 μl of the cDNAs of each sample were used for ordinary PCR to test whether the corresponding primers generated products. Real time PCR was carried out with the SYBR Green PCR Master Mix system (Takara) on an ABI 7500 Real-time PCR platform. The PCR amplification conditions were performed according to Zou et al [21 (link)]. The ΔΔC_t Value for each gene was analyzed in Microsoft Excel and used to indicate the change in expression level between two samples.

In order to verify the accuracy of the sequencing results, 10 differential genes were selected for RT-qPCR analysis. The TransScript One-Step gDNA Removal and cDNA Synthesis Super-Mix Reverse Transcription Kit were used for reverse transcription. Total RNAs extracted from leaves and roots from the control and salt treatment groups were used to synthesize cDNA. Primer Premier 5 software was used to design primers (Supplementary Table S1) (Regina et al., 2010 (link); Bahrini et al., 2011 (link)). Real-time PCR was performed using the Applied Biosystems 7500/7500 fast real-time PCR system (ABI, Foster City, California, USA) and the SYBR Green PCR Master Mix system (Takara). We referred to the Trans-Start Top Green qPCR Super-Mix manual for RT-qPCR with triplicates. The 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to quantitatively calculate the relative expression level of candidate genes.