Light cycle real time pcr machine

Total RNA was extracted from RAW 264.7 cells using RNAios Plus reagent (Takara, Kusatsu, Japan) and RNA concentration was measured with a Nano‐300 ultramicro‐spectrophotometer (All Sheng, Zhejiang, China). TIANScript II RT Kit (KR107‐02, Tiangen, Shanghai, China) was used to synthesize cDNA. qPCR was performed using SYBR Green (11198ES08, YEASEN, Shanghai, China) on a Light Cycle® Real‐Time PCR machine (Roche, Switzerland). The primers used were synthesized by Sangon Biotech (Shanghai, China) (Table S2). GAPDH housekeeping gene was used as internal control. Transcript levels of target genes were calculated using the 2^−∆∆Ct method.

Total RNA was extracted from B16–F10 melanoma cells treated with 8-MOP for 12 h using RNAios Plus reagent (Takara, Kusatsu, Japan). RNA concentration was measured with a Nano300 ultramicro-spectrophotometer (All Sheng, China). M-MLV reverse transcriptase (Promega, China) was used to synthesize cDNA following the manufacturer's protocol. qPCR was performed by using SYBR Green on a Light Cycle® Real-Time PCR machine (Roche, USA). GAPDH gene was used as an internal control for normalization. Transcript levels of target genes were calculated relative to a reference gene using the 2^–ΔΔCt method [24 (link)]. Primers used in this study were synthesized by Sangon Biotech (China) and are shown in Supplementary Table S2.