Reverse transcription (RT)-PCR

RNA of biotransformation enzymes and transporters was detected by reverse transcription (RT)-PCR. Genes were chosen based on their significance in drug metabolism and the availability of sequence data.

The expression of hepatocyte-specific genes in BFH12 cells of passage 20, foetal hepatocytes and adult liver tissue was determined by RT-PCR. Total RNA from BFH12 grown in 25 cm² cell culture flasks was extracted using the ReliaPrep™ RNA Cell MiniPrep System (Promega, Madison, USA). RNA from adult liver tissue was extracted with the ReliaPrep™ RNA Tissue MiniPrep System. Integrity of the RNA extracted from the samples was assessed by gel electrophoresis in an agarose gel stained with ethidium bromide (EtBr). Only intact RNA showing sharp, clear 28S and 18S rRNA bands was used for subsequent cDNA synthesis. Reverse transcription was performed with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, Schwerte, Germany). PCR amplification was carried out on a T1 Thermocycler (Biometra, Göttingen, Germany) using the FastGene^® Optima HotStart Ready Mix (Nippon Genetics, Düren, Germany). Initial denaturation was performed at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s and extension at 72 °C for 1 min. Genes of interest were cytochrome P450 1A1 (CYP1A1), 1A2 (CYP1A2), 2B6 (CYP2B6), 2C19 (CYP2C19), 2E1 (CYP2E1), 3A4 (CYP3A4), UDP glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) and A6 (UGT1A6), glutathione S-Transferase M1 (GSTM1), P-glycoprotein (P-gp), ATP-binding cassette sub-family G member 2 (ABCG2), ATP-binding cassette sub-family C member 1 (ABCG2), organic cation transporter 1 (OCT1), organic anion-transporting polypeptide 1B3 (OATP1B3), and sodium-taurocholate cotransporting polypeptide (NTCP). β-actin (ACTB) served as positive control. The DME genes are designated by conventional (human) nomenclature. A new nomenclature for bovine DMEs has been proposed (Zancanella et al. 2010) which is not yet standardized or implemented in the National Center for Biotechnology Information (NCBI) database. The gene-specific PCR primers were designed from bovine sequence data using the primer-BLAST tool of the NCBI and were synthesised by Metabion International AG (Planegg, Germany). Primer sequences and PCR conditions are given in Table 1. PCR products were analysed by gel electrophoresis (3.5% agarose) and ethidium bromide staining. Product sizes were determined using a 100 bp DNA ladder (Thermo Scientific GeneRuler 100 bp DNA Ladder; Thermo Scientific, Schwerte, Germany). In order to compare the gene expression of BFH12 and primary cells, experiments were also conducted with adult liver tissue and foetal bovine hepatocytes (cryopreserved cells). The cells were cultivated using the same cell culture protocol as for cultivation of BFH12 before cryopreservation. Gene expression analysis of BFH12 cells and foetal hepatocytes was performed in two independent experiments (n = 2).

Primers and conditions for gene expression analysis by RT-PCR