RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, US) and was reverse transcribed to cDNA using a reverse transcription kit (TOYOBO Life Science (Shanghai), Shanghai, China). Real-time PCR reactions were performed using Faststart Universal SYBR Green Master Mix (Roche, Basel, Switzerland) in a real-time Thermocycler (Applied Biosystems, Foster City, CA, US) in triplicate. β-actin was used as an internal control (Forward primer: 5′-GCCGTGGTGGTGAAGCTGT-3′ and reverse primer: 5′-ACCCACACTGTGCCCATCTA-3′). The forward primer for detection of CYP27B1 was 5′-ACGGTGTCCAACACGCTCT-3′ and the reverse primer was 5′-AACAGTGGCTGAGGGGTAGG-3′. Data are presented as relative mRNA levels calculated by the 2−ΔCt(ΔCt = Ct of target gene minus Ct of β-actin) (Livak & Schmittgen, 2001).

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