M5 hiper real time pcr super mix with low rox

FastStart Universal SYBR Green Master Mix (ROX) on an ABI 7500 Real-Time PCR System was used to amplify cDNA through quantitative real-time PCR. The amplification reactions contained 2 µL of diluted RT product, 0.5 µL of objective mRNA forward primer, 0.5 µL of objective mRNA reverse primer, 10 µL of 2× M5 HiPer Real-time PCR Super mix with Low Rox (Mei5 Biotechnology, MF797, Beijing, China), and 7 µL RNase-free water. The PCR cycling conditions were 95 °C for 5 min followed by 40 cycles at 95 °C for 20 s and 60 °C for 50 s. MC1R expression was normalized to human β-actin expression, and the experiment was performed in triplicate. By means of the ∆∆Ct equation, the expression of MC1R in clinical tissue samples was calculated in comparison with the endogenous control β-actin. Related primers are shown below: MC1R, 5′-GCTACCACAGCATCGTGACC-3′ (forward primer) and 5′-ACGTGGTCGTAGTAGGCGAT-3′ (reverse primer); β-actin, 5′-CATGTACGTTGCTATCCAGGC-3′ (forward primer) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse primer).

For single-cell RT-qPCR, cDNA from individual cells was diluted with water in a ratio of 1:20. RT-qPCR was performed using 2× M5 HiPer Realtime PCR Super mix with Low Rox (Mei5 Biotechnology, MF797) on a Roche LightCycler^® 480 Instrument II. For bulk-cell RT-qPCR, total RNA was extracted from the collected cells using the RNAprep Pure Micro kit (Tiangen, DP420), and reverse transcription was performed with HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-1). Primer sequences are listed in Supplementary information, Table S9.