Qpcr master mix

Total RNA from cells was isolated using Trizol reagent (Invitrogen) according to manufacturer's guidelines. Equal amounts of RNA were reverse-transcribed using M-MuLV Reverse Transcriptase (NEB) and real-time PCR analysis was performed using qPCR Master-Mix (75 mM Tris-HCl, pH 8.8, 20 mM (NH₄)₂SO₄, 3 mM MgCl₂, 0.3 M Trehalose (all from Roth, Karlsruhe, Germany), 0.01% Tween-20, 0.25% Triton X-100 (both from Applichem, Darmstadt, Germany), SYBR Green 1:80 000 (Fermentas, Schwerte, Germany), 0.2 mM dNTPs, 20 U/ml Taq-polymerase (both from Primetech, Minsk, Belarus) and 0.3 mM primers. Primers for total UGT1A: 5′-ATCTGCTTGGTCACCCGATG-3′ and 5′-TCCATGCGCTTTGCATTGTC-3′ for UGT1A1: 5′-GCCATTCCAAAGGGAGGATGTG-3′ and 5′-TGGGAACAGCCAGACAAAAGC-3′ for cluster UGT1A3-5: 5′-CATAATGAGGCCCTGATCAGGC-3′ and 5′-AATCGACAGGTACTTAGCCAG-3′ for UGT1A6: 3′-GCTGGTGGTCCCTCAGGAC-5′ and 5′-CAGCTCTTCTTGGTCATACGGC-3′ for cluster UGT1A7-10: 5′-CACAGTGCCCTGCTCCTC-3′ and 5′-GTTTGGAGAATTTCAGAGGCTATTTC-3′ for normalizer HPRT1: 5′-ATGCTGAGGATTTGGAAAGG-3′, and 5′-TCATCACATCTCGAGCAAGAC-3′. Primers (Metabion, Mansried, Germany) were used in a two-step protocol (2 min at 95 °C pre-heating; 40 cycles at 95 °C for 15 s followed by 58 °C for 1 min).