Transscript first strand cdna synthesis kit

DMEM/F12, penicillin-streptomycin, fetal bovine serum, TRIzol reagent, SYBR Green, and RIPA buffer were supplied from Thermo Fisher Scientific (Waltham, MA, USA). Epidermal growth factor, and ITS (the mixture of insulin, transferrin, and selenious acids) were obtained from Corning Incorporated (New York, NY, USA), dimethyl sulfoxide, and genistein (synthetic product, purity ≥ 98%) were provided by Sigma-Aldrich (St. Louis, MO, USA). ROS assay kit was obtained from Beyotime Biotechnology (Shanghai, China). The cell counting kit (CCK-8) was bought from Med Chem Expression (Princeton, NJ, USA). The malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) assay kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Trans Script First-Strand cDNA Synthesis Kit was supplied by Trans Gen Biotech (Beijing, China). The VDF membranes, and ECL agent were provided by Bio-Rad Laboratories, Incorporated (Irvine, CA, USA).

Total RNA of the cotton leaves and roots was isolated using the Plant Total RNA Extraction Kit (Sangon Biotech, Shanghai, China) according to the instruction manual. Two micrograms of RNA was reverse transcribed for first strand cDNA synthesized following the manufacturer’s protocol using the TransScript First-Strand cDNA Synthesis kit (TransGen).
According to the protocols of the Minimum Information for Publication of Quantitative Real Time PCR Experiments (Bustin et al., 2009 (link)), diluted cDNA was used for qPCR with SYBR green using the CFX96 Touch^TM Real-Time PCR detection systems (Bio-Rad, Foster City, CA, United States). All gene expression was calculated using the dCt or ddCt algorithm. To normalize the gene expression, the UB7 gene was used as an internal standard. All the gene specific primers involved in this study were listed in Supplementary Table S1.

Total RNA was extracted from the brains of immature turbot using the RNA Isolation Kit (Catalog No. 220011) (Fastagen Biotech, China). Quality and concentration of RNA were measured using 1.0% agarose gels electrophoresis and assessed by spectrophotometry (ND-2000, Nanodrop, USA). The first-strand cDNA was synthesized using a TransScript First-Strand cDNA synthesis kit (AT301-02) (Transgen, China) following the manufacturer’s instructions. Subsequently, cDNA fragments of turbot kiss1, kiss2, kissr2, and kissr3 were amplified with primers (Table 1). Finally, 5’-RACE and 3’-RACE were performed for full-length cDNAs using the SMARTer RACE cDNA Amplification Kit (Catalog No.634923) (Clontech, USA) with gene-specific primers (GSPs) (Table 1). The diluted first-round PCR products were used as templates for nested PCR with corresponding primers of GSP-nest (Table 1). All PCR programs were performed using a PTC-100 thermal cycle (Bio-Rad, USA). PCR was performed in a 25 μl reaction volume containing 1 μl of diluted cDNA, 1 μl of each primer (10 μM), 12.5 μl of Premix buffer (with dNTPs), and 9.5 μl ddH₂O, with amplification procedure of denaturation at 94°C for 5minutes; 35 cycles of amplification at 94°C for 30 seconds, annealing with specific temperature of each primer (Table 1) for 45 seconds, elongation with 72°C for 30 seconds, an additional elongation at 72°C for 10 minutes.