Real-time reverse transcriptase-polymerase chain reaction
To measure the mRNA expression levels of various CYPs, the cells were either treated with 500 μM MA and/or transfected with plasmid encoding gp120. Upon termination of the treatment, total RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA (120 ng) was reverse transcribed into cDNA at 37 °C for 60 min using a 2-step TaqMan Gene Expression Kit (Applied Biosystems, Foster City, CA, USA). The cDNA obtained was amplified for GAPDH (4333764F), CYP1A1 (Hs01054794_m1), CYP2B6 (Hs03044636_m1), CYP2E1 (Hs00559367_m1), CYP3A4 (HS00430021_m1), CYP2D6 (Hs00164385_m1), and CYP2A6 (Hs0071162_m1) using the probe mix obtained from Applied Biosystems as per the manufacturer's protocol. The expression values for various CYPs were normalized using GAPDH as a housekeeping gene. Relative fold expressions for various genes were analyzed using the 2−ΔΔCt method.