Sybr green master mix

To determine the expression levels of GhSOS1 in cotton seedlings treated with various abiotic stresses and analyze the expression of GhSOS1 and the salt-related genes in the transgenic Arabidopsis plants, total RNA from cotton seedlings and Arabidopsis plants was respectively isolated from the collected tissues using TRIzol reagent (Aidlab Biotech). The amplification of quantitative real-time PCR products was performed in a reaction mixture of 20 μL of SYBR Green Master Mix (Transgen Biotech) according to the manufacturer’s instructions. Three biological replicates and three technical replicates for each sample were performed. The primers used for quantitative real-time PCR are shown in S1 Table.

Total RNAs were prepared from cells or kidney tissues by using ultra-pure TRIzol reagent (Tiangen) according to the manufacturer’s instructions. Reverse transcription was performed on equal amounts of total RNA (2.5 μg) by using random hexanucleotide primers to produce a cDNA library for each sample. qPCR were run in the ABI ViiA™7 Real-Time System (Life Technologies) by using SYBR Green Master Mix (Transgen, Beijing, China) and gene-specific primers (Table S1 online). Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2^−ΔΔCt) compared with lean controls.

The qRT-PCR assays were performed using the LightCycler® 480 instrument II (Roche Life Science, Switzerland) with SYBR Green Master Mix (Transgen Biotech, Beijing, China). All the qPCR primer pairs (for primer sequences, see Table S1, see online supplementary material) displayed similar amplification efficiency (90–110%). Relative quantities were normalized against the geometric mean of the reference genes VvACTIN and VvGAPDH using the 2^-δδCt method [66 (link)].

Total RNA was isolated from the muscle tissue using TRIzol reagent (TransGen Biotech, Beijing, China). The integrity, purity, and concentration of RNA were determined by 1% agarose gel electrophoresis and nucleic acid/protein analyzer. cDNA was synthesized with a commercial RT Master Mix kit (TransGen). qPCR amplification was performed with the BioRad PCR machine with SYBR green master mix (TransGen) following the manufacturer's guidelines. All samples were analyzed in triplicates. The optimal annealing temperature of each primer was determined. The correlation coefficients of all the standard curves were >0.99, and the amplification efficiency values were between 90 and 110% (3.6>slope>3.1). The specificity of amplification was determined by the melting curve analysis at the end of the target gene amplification. The relative mRNA expression of target genes was determined after normalization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference using the 2^−ΔΔCt method (25 (link)). The primer sequences of the target genes and GAPDH were designed by Primer 5.0 software (Premier Biosoft, Palo Alto, CA, United States) and validated by BLAST sequence alignment in NCBI (Table 3).

The total RNA was extracted by using the RNeasy system (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, and the RNA was reversed transcribed into cDNA. The PCR reactions for the MYCN, MYC, MYCL, EZH2, HBG1, KLF1, p21 and GAPDH genes were performed using the ABI PRISM 7500 System (Applied Biosystems, Foster city, CA, USA) and SYBR Green Master Mix (Transgene, Beijing, China). The relative expression was calculated using 2Δ-ΔCT.