RNA Extraction and qRT-PCR Analysis

SIM A-9 cells were collected from plates and total RNA was extracted using a Quick-RNA Miniprep Kit (ZYMO Research, Irvine, CA, United States). 1 μg of total RNA was subjected to reverse transcription using M-MLV Reverse Transcriptase (Thermo Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. For qRT-PCR, PowerUp SYBR Green Master Mix (Thermo Scientific) was used, and experiments conducted on a Bio-Rad iQ5 thermal cycler (Bio-Rad Laboratories, Hercules, CA, United States). Differences in expression were evaluated by the comparative cycle threshold method using GAPDH as a control. To extract total RNA from mouse tissues, TriZol reagent (Life Technologies) was added to homogenized tissue according to the manufacturer’s instructions followed by qRT-PCR assays as we described previously (Qiu et al., 2020 (link)).