Hifair 3 first strand cdna synthesis supermix kit

Following the instructions, RNeasy kits (Qiagen, German) were used to extract total RNA from cells. After obtaining and quantifying RNA, reverse transcription was performed using the Hifair^® Ⅲ first Strand cDNA Synthesis SuperMix kit (YEASEN, China). After cDNA was obtained, the relative quantification of the target gene in cDNA can be completed with the Hieff^® qPCR SYBR Green Master Mix kit (YEASEN, China). GAPDH was used as an internal reference for quantitative calculation. All procedures were performed on Bio-Rad CFX96 Connect and Roche 480Ⅱ instrument. The primer sequences required for quantitative PCR were presented as follows: GAPDH, forward 5′-ACC​CAG​AAG​ACT​GTG​GAT​GG-3′ and reverse 5′-CAC​ATT​GGG​TAG​GAA​CAC-3′; Cathepsin K, forward 5′-CTT​CCA​ATA​CGT​GCA​GCA​GA-3′ and reverse 5′-TCT​TCA​GGG​CTT​TCT​CGT​TC-3′; CTR, forward 5′-TGC​AGA​CAA​CTC​TTG​GTT​GG-3′ and reverse 5′-TCG​GTT​TCT​TCT​CCT​CTG​GA-3′; TRAP, forward 5′-CTG​GAG​TGC​ACG​ATG​CCA​GCG​ACA-3′ and reverse 5′-TCC​GTG​CTC​GGC​GAT​GGA​CCA​GA-3′; NFATc1, forward 5′-CCG​TTG​CTT​CCA​GAA​AAT​AAC​A-3′ and reverse 5′-TGT​GGG​ATG​TGA​ACT​CGG​AA-30′; V-ATPase d2, forward 5′-AAG​CCT​TTG​TTT​GAC​GCT​GT-3′ and reverse 5′-TTC​GAT​GCC​TCT​GTG​AGA​TG-3′; V-ATPase a3, forward 5′-TGG​CTA​CCG​TTC​CTA​TCC​TG-3′ and reverse 5′-CTT​GTC​CGT​GTC​CTC​ATC​CT-3′;

Total RNA was extracted from the retina of each group using the Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, United States). Subsequently, RNA was re-verse-transcribed to cDNA using Hifair® Ⅲ first Strand cDNA Synthesis SuperMix kit (Yeasen Biotechnology Co. Ltd., Shanghai, China) following the manufacturer’s instructions. The reaction of real-time PCR was performed in Hieff ® qPCR SYBR Green Master Mix (Yeasen Biotechnology Co. Ltd., Shanghai, China), which contained cDNA 1.2 µL (800 ng/μL), SYBR green mix 10 μL, 0.4 µL each forward or reverse primer (2 μmol L⁻¹), 8 µL distilled water. The primer pairs were designed by Beacon Designer 7 as per the published gene sequences of the NCBI database. Real-time PCR was performed using a System 7,500 instrument (Applied Biosystems, Carlsbad, CA, United States), and the following thermocycling conditions were as follows: 95°C pre-denaturation for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 35 s, 95°C for 15 s. After the reactions, melt curve analysis was used to assess whether the intercalating dye qPCR assays had produced single, specific products. Relative expression of target genes was normalized to β-actin and calculated according to the 2^−∆∆Ct method (Livak and Schmittgen, 2001 (link)). Each sample was analyzed in triplicate. All primers are listed in Supplementary Table S5.