Cfx manager 2.1 detection system

The cells were collected with TRIzol (TaKaRa, Japan). Total RNA was extracted, and reverse transcription was performed using PrimeScript RT Master Mix (TaKaRa, Japan). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed in a Bio-Rad CFX Manager 2.1 Detection System (Bio-Rad) using Real MasterMix (SYBR Green) (Tiangen Biotech, China). RT-qPCR amplification was performed in duplicate (the program was as follows: denaturation at 95°C for 15 minutes followed by 40 cycles of 95°C for 10 seconds and 60°C for 40 seconds). All primer sequences are listed in Table 1. The messenger RNA (mRNA) expression was normalized to that of Gapdh.

Isolation of total RNA and real-time quantitative PCR (RT-qPCR) were performed following our previous methods.³⁵ (link) Total RNA was extracted form ARPE-19 cells with TRIeasy LS Total RNA Extraction Reagent (19201ES60; Yeasen, Shanghai, China). The cDNA Synthesis SuperMix (11120ES60, Yeasen; Shanghai, China) was used to perform reverse transcription. SYBR Green master mix (11201ES03, Yeasen; Shanghai, China) was used to conduct RT-qPCR in Bio-Rad CFX Manager 2.1 Detection system (Hercules, CA, USA). The information of human primers is listed in Table 2.