Taq purple mix 2

Total RNA was extracted with Trizol (Life Technologies) and reverse transcribed with M-Maxima™ First Strand cDNA Synthesis Kit (ThermoFisher). PCR was performed with Taq’Ozyme Purple Mix 2 (Ozyme, Saint-Cyr-l’Ecole, France) (OZYA007-200XL). The sequence of primers for CD46 were: F: 5’-GTGGTCAAATGTCGATTTCCAGTAGTCG-3’, R: 5’-CAAGCCACATTGCAATATTAGGTAAGCCACA-3’^{41 (link)}.

The CYP51A, CYP51B, and CYP51C genes were amplified using previously described primers [27 (link)]. The reaction mixtures contained 0.2 µM of each primer, 25 µL of Taq’Ozyme Purple Mix 2 (Ozyme, Saint-Cyr-l’École, France), and 5 µL of gDNA in a final volume of 50 µL. Samples were amplified using a CFX96 Real-Time PCR System (BioRad, Marnes-la-Coquette, France) and the following cycling protocol: one initial cycle of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 2 min at 72 °C, with one final cycle of 5 min at 72 °C. PCR quality was assessed by 1.5% agarose gel electrophoresis (120 V, 20 min) and visualization using the Fusion Fx7 device (Vilber Lourmat, Eberhardzell, Germany). PCR product sequencing was performed using the Sanger method by Eurofins Genomics (Ebersberg, Germany) with the same primers.