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Real-time RT-PCR normalisation; strategies and considerations

Abstract

Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.

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References

  1. Atamas SP . Alternative splice variants of cytokines: making a list. Life Sci 1997; 61: 1105–1112.

    CAS  Article  Google Scholar 

  2. Demissie A, Abebe M, Aseffa A et al. Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2. J Immunol 2004; 172: 6938–6943.

    CAS  Article  Google Scholar 

  3. Fletcher HA, Owiafe P, Jeffries D et al. Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4delta2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation. Immunology 2004; 112: 669–673.

    CAS  Article  Google Scholar 

  4. Bustin SA, Nolan T . Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004; 15: 155–166.

    PubMed  PubMed Central  Google Scholar 

  5. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A . Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 2004; 37: 112–119.

    CAS  Article  Google Scholar 

  6. Bustin SA . Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002; 29: 23–39.

    CAS  Article  Google Scholar 

  7. Stahlberg A, Kubista M, Pfaffl M . Comparison of reverse transcriptases in gene expression analysis. Clin Chem 2004; 50: 1678–1680.

    CAS  Article  Google Scholar 

  8. Spanakis E . Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls. Nucleic Acids Res 1993; 21: 3809–3819.

    CAS  Article  Google Scholar 

  9. Hansen MC, Nielsen AK, Molin S, Hammer K, Kilstrup M . Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels. J Bacteriol 2001; 183: 4747–4751.

    CAS  Article  Google Scholar 

  10. Talaat AM, Howard ST, Hale W, Lyons R, Garner H, Johnston SA . Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis. Nucleic Acids Res 2002; 30: e104.

    Article  Google Scholar 

  11. Rocha EP . The replication-related organization of bacterial genomes. Microbiology 2004; 150 (Part 6): 1609–1627.

    CAS  Article  Google Scholar 

  12. Tanaka S, Furukawa T, Plotkin SA . Human cytomegalovirus stimulates host cell RNA synthesis. J Virol 1975; 15: 297–304.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Piechaczyk M, Blanchard JM, Marty L et al. Post-transcriptional regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression in rat tissues. Nucleic Acids Res 1984; 12: 6951–6963.

    CAS  Article  Google Scholar 

  14. Stout JT, Chen HY, Brennand J, Caskey CT, Brinster RL . Expression of human HPRT in the central nervous system of transgenic mice. Nature 1985; 317: 250–252.

    CAS  Article  Google Scholar 

  15. Blomberg J, Andersson M, Faldt R . Differential pattern of oncogene and beta-actin expression in leukaemic cells from AML patients. Br J Haematol 1987; 65: 83–86.

    CAS  Article  Google Scholar 

  16. Barbu V, Dautry F . Northern blot normalization with a 28S rRNA oligonucleotide probe. Nucleic Acids Res 1989; 17: 7115.

    CAS  Article  Google Scholar 

  17. Bas A, Forsberg G, Hammarstrom S, Hammarstrom ML . Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 2004; 59: 566–573.

    CAS  Article  Google Scholar 

  18. Bemeur C, Ste-Marie L, Desjardins P et al. Decreased beta-actin mRNA expression in hyperglycemic focal cerebral ischemia in the rat. Neurosci Lett 2004; 357: 211–214.

    CAS  Article  Google Scholar 

  19. Schmid H, Cohen CD, Henger A, Irrgang S, Schlondorff D, Kretzler M . Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int 2003; 64: 356–360.

    CAS  Article  Google Scholar 

  20. Schulz WA, Eickelmann P, Hallbrucker C, Sies H, Haussinger D . Increase of beta-actin mRNA upon hypotonic perfusion of perfused rat liver. FEBS Lett 1991; 292: 264–266.

    CAS  Article  Google Scholar 

  21. Tricarico C, Pinzani P, Bianchi S et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 2002; 309: 293–300.

    CAS  Article  Google Scholar 

  22. Ullmannova V, Haskovec C . The use of housekeeping genes (HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR. Folia Biol (Praha) 2003; 49: 211–216.

    CAS  Google Scholar 

  23. Koch I, Weil R, Wolbold R et al. Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab Dispos 2002; 30: 1108–1114.

    CAS  Article  Google Scholar 

  24. Dheda K, Huggett JF, Kim LU, Zumla A . Type 2 cytokines in respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med 2004; 169: 1167–1168.

    Article  Google Scholar 

  25. Winer J, Jung CK, Shackel I, Williams PM . Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999; 270: 41–49.

    CAS  Article  Google Scholar 

  26. Vandesompele J, De Preter K, Pattyn F et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034.

    Article  Google Scholar 

  27. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26: 509–515.

    CAS  Article  Google Scholar 

  28. Andersen CL, Jensen JL, Orntoft TF . 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 2004; 64: 5245–5250.

    CAS  Article  Google Scholar 

  29. Cronin M, Ghosh K, Sistare F, Quackenbush J, Vilker V, O'Connell C . Universal RNA reference materials for gene expression. Clin Chem 2004; 50: 1464–1471.

    CAS  Article  Google Scholar 

Download references

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Correspondence to J Huggett.

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Huggett, J., Dheda, K., Bustin, S. et al. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6, 279–284 (2005). https://doi.org/10.1038/sj.gene.6364190

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  • DOI: https://doi.org/10.1038/sj.gene.6364190

Keywords

  • qPCR
  • real-time RT-PCR
  • housekeeping genes
  • normalisation
  • reference gene

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