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Analysis of CYP2C9 mRNA levels by quantitative real-time PCR

FF Ferenc Fekete
KM Katalin Mangó
MD Máté Déri
EI Evelyn Incze
AM Annamária Minus
KM Katalin Monostory
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Total RNA (3 μg) was reverse-transcribed into single-stranded cDNA by using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Wilmington, DE). The real-time PCR with human cDNA was performed by using KAPA Fast Probes Mastermix (KAPA Biosystems, Cape Town, South Africa) and TaqMan probes for CYP2C9 (BioSearch Technologies, Novato, CA). The quantity of the target RNA relative to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined. GAPDH expression is constant in all cells and independent from experimental conditions; therefore, its expression was set to 1, and CYP2C9 mRNA levels were normalized by GAPDH expression. The sequences of primers and probes used for the real-time PCR analyses of CYP2C9 and GAPDH expression were previously reported by Déri et al. (2020)30.

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