Sybr green no rox

Total RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN) with on-column DNase I treatment. RNA quantity and purity were determined by spectrophotometry. RNA was reverse-transcribed into cDNA using SuperScript III VILO reverse transcriptase (Life Technologies) and diluted with UltraPure dH₂O (Life Technologies). qPCR reactions comprised 1X SYBR Green No-Rox (Bioline), 250 nM forward and reverse primers (Supplementary Table 1), and 2 µL diluted cDNA up to a final volume of 10 µL. Cycling and analysis were conducted using a Mic qPCR Cycler (BioMolecular Systems) with the following conditions: 95°C for 10 min, and 35 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. Melt curve analysis was used to confirm specific amplification of targets. Relative mRNA expression levels were calculated using the comparative Ct method, normalised to β-actin (ACTB).