Sybr green rt pcr kit

TRIzol reagent (Invitrogen, USA) was used to extract total RNA from the cells, and the cDNA synthesis kit (Takara, Japan) was used to reverse-transcribe the extracted total RNA into cDNA in accordance with the kit’s instructions. SYBR Green RT-PCR Kits (Takara, Japan) were used for the qPCR, and 2^−ΔΔCt was used to determine the relative mRNA expression. β-actin provided internal control. Table 1 contained a list of the primers.

Total RNA was extracted from cells utilizing RNeasy kits (Beyotime, China) and reverse transcribed with one-step RT-PCR kits (Beyotime, China) at 37°C lasting 30 min, in accordance with the manufacturer’s protocols. qPCR was conducted utilizing SYBR Green RT-PCR kits (Takara, China). The thermocycling conditions were as follows: 95°C lasting 5 min; 40 cycles of 95°C lasting 40 s, 60°C lasting 30 s, and 72°C lasting 30 s. The following primers were used for PCR: AKR1B1: 5′-TTT​TCC​CAT​TGG​ATG​AGT​CGG-3′ (forward), 5′-CCT​GGA​GAT​GGT​TGA​AGT​TGG-3′ (reverse); CPVL: 5′-TGG​AAG​GTG​ATT​GTT​TCG​CTG-3′ (forward), 5′-GTC​TCC​CTT​AGG​TGG​CAT​GGA-3′ (reverse); CTSL: 5′- CTT​TTG​CCT​GGG​AAT​TGC​CTC -3′ (forward), 5′-CAT​CGC​CTT​CCA​CTT​GGT​C-3′ (reverse); and GAPDH: 5′-GGA​GCG​AGA​TCC​CTC​CAA​AAT-3′ (forward), 5′-GGC​TGT​TGT​CAT​ACT​TCT​CAT​GG-3′ (reverse). The fold change in mRNA expressions was determined with the 2^−ΔΔCq method.