Skip to main content

Advertisement

SpringerLink
Log in

Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data

  • Original Article
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Abstract

Suitable reference genes for correct quantification of reverse transcription PCR (qRT-PCR) have to be constantly expressed in all samples under investigation. Thus, it is mandatory to determine expression stability of control genes before normalization. We aimed to establish optimum inducing concentrations for the prototypical enzyme and drug transporter inducer rifampicin in LS180 cells and concurrently assessed reference gene stability under rifampicin treatment. LS180 cells were treated with increasing concentrations of rifampicin (up to 200 μM), and expression of eight different reference genes and some target genes (CYP3A4, ABCB1, and ABCC1) was quantified using real-time qRT-PCR. To check whether the results can be generalized, HepG2 cells were also investigated. We demonstrated that higher concentrations of rifampicin (>50 μM) change the expression of reference genes and thus may complicate and adulterate normalization of qRT-PCR data. The results stress the need for proper validation of potential reference genes in respective cells, tissues, and particular experimental conditions. Programs like geNorm and NormFinder alone do not warrant an adequate choice of the most suitable reference gene. Scrutiny of the reference gene expression and plausibility of the data remain necessary and protect from erroneous quantification and misinterpretation of qRT-PCR data.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Albermann N, Schmitz-Winnenthal FH, Z’graggen K, Volk C, Hoffmann MM, Haefeli W, Weiss J (2005) Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver. Biochem Pharmacol 70:949–958

    Article  PubMed  CAS  Google Scholar 

  • Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250

    Article  PubMed  CAS  Google Scholar 

  • Ayed-Boussema I, Pascussi JM, Maurel P, Bacha H, Hassen W (2011) Effect of aflatoxin B1 on nuclear receptors PXR, CAR, and AhR and their target cytochromes P450 mRNA expression in primary cultures of human hepatocytes. Int J Toxicol 31:86–93

    Article  PubMed  Google Scholar 

  • Buss WC, Morgan R, Guttmann J, Barela T, Stalter K (1978) Rifampicin inhibition of protein synthesis in mammalian cells. Science 200:432–434

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  PubMed  CAS  Google Scholar 

  • Cerveny L, Svecova L, Anzenbacherova E, Vrzal R, Staud F, Dvorak Z, Ulrichova J, Anzenbacher P, Pavek P (2007) Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways. Drug Metab Dispos 35:1032–1041

    Article  PubMed  CAS  Google Scholar 

  • Gutmann H, Fricker G, Török M, Michael S, Beglinger C, Drewe J (1999) Evidence for different ABC-transporters in Caco-2 cells modulating drug uptake. Pharm Res 16:402–407

    Article  PubMed  CAS  Google Scholar 

  • Harmsen S, Koster AS, Beijnen JH, Schellens JH, Meijerman I (2008) Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction. Drug Metab Dispos 36:1166–1171

    Article  PubMed  CAS  Google Scholar 

  • Hartmann GR, Heinrich P, Kollenda MC, Skrobranek B, Tropschug M, Weiß W (1985) Molecular mechanism of action of the antibiotic rifampicin. Angew Chem Int Ed Engl 24:1009–1014

    Article  Google Scholar 

  • Kirby BJ, Collier AC, Kharasch ED, Dixit V, Desai P, Whittington D, Thummel KE, Unadkat JD (2011) Complex drug interactions of HIV protease inhibitors 2: in vivo induction and in vitro to in vivo correlation of induction of cytochrome P450 1A2, 2B6, and 2C9 by ritonavir or nelfinavir. Drug MetabDispos 39:2329–2337

    CAS  Google Scholar 

  • König SK, Herzog M, Theile D, Zembruski N, Haefeli WE, Weiss J (2010) Impact of drug transporters on cellular resistance towards saquinavir and darunavir. J Antimicrob Chemother 65:2319–2328

    Article  PubMed  Google Scholar 

  • Lundby C, Nordsborg N, Kusuhara K, Kristensen KM, Neufer PD, Pilegaard H (2005) Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. Eur J Appl Physiol 95:351–360

    Article  PubMed  CAS  Google Scholar 

  • Martin P, Riley R, Back DJ, Owen A (2008) Comparison of the induction profile for drug disposition proteins by typical nuclear receptor activators in human hepatic and intestinal cells. Br J Pharmacol 153:805–819

    Article  PubMed  CAS  Google Scholar 

  • Medoff G, Kwan CN, Schlessinger D, Kobayashi GS (1973) Potentiation of rifampicin, rifampicin analogs, and tetracycline against animal cells by amphotericin B and polymyxin B. Cancer Res 33:1146–1149

    PubMed  CAS  Google Scholar 

  • Nibourg GA, Huisman HT, van der Hoeven TV, van Gulik TM, Chamuleau RA, Hoekstra R (2010) Stable overexpression of pregnane X receptor in HepG2 cells increases its potential for bioartificial liver application. Liver Transpl 16:1075–1085

    Article  PubMed  Google Scholar 

  • Niemi M, Backman JT, Fromm MF, Neuvonen PJ, Kivistö KT (2003) Pharmacokinetic interactions with rifampicin: clinical relevance. Clin Pharmacokinet 42:819–850

    Article  PubMed  CAS  Google Scholar 

  • Nishimura M, Koeda A, Suzuki E, Shimizu T, Kawano Y, Nakayama M, Satoh T, Narimatsu S, Naito S (2006a) Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes. Biochem Biophys Res Commun 346:1033–1039

    Article  PubMed  CAS  Google Scholar 

  • Nishimura M, Koeda A, Suzuki E, Kawano Y, Nakayama M, Satoh T, Narimatsu S, Naito S (2006b) Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes. Drug Metab Pharmacokinet 21:297–307

    Article  PubMed  CAS  Google Scholar 

  • Peters T, Lindenmaier H, Haefeli WE, Weiss J (2006) Interaction of the mitotic kinesin Eg5 inhibitor monastrol with P-glycoprotein. Naunyn Schmiedebergs Arch Pharmacol 372:291–299

    Article  PubMed  CAS  Google Scholar 

  • Pogo BG (1972) Specific inhibition by rifampicin of transcription in human lymphocytes stimulated by phytohemagglutinin. J Cell Biol 55:515–519

    Article  PubMed  CAS  Google Scholar 

  • Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862

    Article  PubMed  Google Scholar 

  • Rae JM, Johnson MD, Lippman ME, Flockhart DA (2001) Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays. J Pharmacol Exp Ther 299:849–857

    PubMed  CAS  Google Scholar 

  • Rhinn H, Scherman D, Escriou V (2008) One-step quantification of single-stranded DNA in the presence of RNA using Oligreen in a real-time polymerase chain reaction thermocycler. Anal Biochem 372:116–118

    Article  PubMed  CAS  Google Scholar 

  • Schaefer O, Ohtsuki S, Kawakami H, Inoue T, Liehner S, Saito A, Sakamoto A, Ishiguro N, Matsumaru T, Terasaki T, Ebner T (2012) Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes. Drug Metab Dispos 40:93–103

    Article  PubMed  CAS  Google Scholar 

  • Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81

    Article  PubMed  CAS  Google Scholar 

  • Suzuki T, Higgins PJ, Crawford DR (2000) Control selection for RNA quantitation. Biotechniques 29:332–337

    PubMed  CAS  Google Scholar 

  • Templeton IE, Houston JB, Galetin A (2011) Predictive utility of in vitro rifampin induction data generated in fresh and cryopreserved human hepatocytes, Fa2N-4, and HepaRG cells. Drug Metab Dispos 39:1921–1929

    Article  PubMed  CAS  Google Scholar 

  • Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291–295

    Article  PubMed  CAS  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F, (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034

    Google Scholar 

  • Weiss J, Herzog M, König S, Storch CH, Ketabi-Kiyanvash N, Haefeli WE (2009) Induction of multiple drug transporters by efavirenz. J Pharmacol Sci 109:242–250

    Article  PubMed  CAS  Google Scholar 

  • Weiss J, Herzog M, Haefeli WE (2011) Differential modulation of the expression of important drug metabolising enzymes and transporters by endothelin-1 receptor antagonists ambrisentan and bosentan in vitro. Eur J Pharmacol 660:298–304

    Article  PubMed  CAS  Google Scholar 

  • Xu C, Li CY, Kong AN (2005) Induction of phase I, II and III drug metabolism/transport by xenobiotics. Arch Pharm Res 28:249–268

    Article  PubMed  CAS  Google Scholar 

  • Yasuda K, Ranade A, Venkataramanan R, Strom S, Chupka J, Ekins S, Schuetz E, Bachmann K (2008) A comprehensive in vitro and in silico analysis of antibiotics that activate pregnane X receptor and induce CYP3A4 in liver and intestine. Drug Metab Dispos 36:1689–1697

    Article  PubMed  CAS  Google Scholar 

  • Zisowsky J, Koegel S, Leyers S, Devarakonda K, Kassack MU, Osmak M, Jaehde U (2007) Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells. Biochem Pharmacol 73:298–307

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Eli Lilly Company (Bad Homburg, Germany) for LY335979. Moreover, they thank Corina Mueller, Stephanie Rosenzweig, and Dominik Menger for excellent technical assistance and Amir Abdollahi, Christian Schwager, and Christiane Rutenberg for the access to and the help with the luminometer.

Author information

Authors and Affiliations

  1. Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

    Johanna Weiss, Dirk Theile & Walter Emil Haefeli

Authors
  1. Johanna Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk Theile
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Walter Emil Haefeli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Johanna Weiss.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(JPEG 59 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weiss, J., Theile, D. & Haefeli, W.E. Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data. Naunyn-Schmiedeberg's Arch Pharmacol 385, 1025–1034 (2012). https://doi.org/10.1007/s00210-012-0782-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00210-012-0782-7

Keywords

Search

Navigation