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Total RNA was synthesized into full-length cDNA by Hifair IV reverse transcriptase (11112ES92, Yeasen) and detected by qPCR using specific primer sequences and Hieff UNICON ColorGPS qPCR SYBR Green Master Mix (11189ES08, Yeasen)

Considering that carotid intima-media thickness (CIMT) is a critical marker of early atherosclerotic lesions, the positive correlation between elevated levels of BCAAs and increased CIMT suggests a potential role for BCAAs in the early development of atherosclerosis (53)

2022;8:e08805

Doctor requested bloodwork that came back normal

(Sato et al., 2011)

Then, the last extension was performed at 72C for 5 min