Complementary Techniques

Validation of Gene Expression Data by Quantitative Real Time PCR
  • Maurizio Provenzano
  • Simone Mocellin
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 593)


Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.


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  1. 1.
    Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet 1999;21:33–37.PubMedCrossRefGoogle Scholar
  2. 2.
    de Arruda M, Lyamichev VI, Eis PS et al. Invader technology for DNA and RNA analysis: Principles and applications. Expert Rev Mol Diagn 2002;2:487–496.PubMedCrossRefGoogle Scholar
  3. 3.
    hang L, Zhou W, Velculescu VE et al. Gene expression profiles in normal and cancer cells. Science 1997;276:1268–1272.CrossRefGoogle Scholar
  4. 4.
    Heid CA, Stevens J, Livak KJ et al. Real time quantitative PCR. Genome Res 1996;6:986–994.PubMedCrossRefGoogle Scholar
  5. 5.
    Klein D. Quantification using real-time PCR technology: Applications and limitations. Trends Mol Med 2002;8:257–260.PubMedCrossRefGoogle Scholar
  6. 6.
    Higuchi R, Dollinger G, Walsh PS et al. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 1992;10:413–417.PubMedCrossRefGoogle Scholar
  7. 7.
    Lee LG, Connell CR, Bloch W et al. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993;21:3761–3766.PubMedCrossRefGoogle Scholar
  8. 8.
    Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5′ Nuclease assays. Curr Opin Biotechnol 1998;9:43–48.PubMedCrossRefGoogle Scholar
  9. 9.
    Didenko W. DNA probes using fluorescence resonance energy transfer (FRET): Designs and applications. Biotechniques 2001;31:1106–1116.PubMedGoogle Scholar
  10. 10.
    Wittwer CT, Herrmann MG, Gundry CN et al. Real-time multiplex PCR assays. Methods 2001;25:430–442.PubMedCrossRefGoogle Scholar
  11. 11.
    Ginzinger DG. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Exp Hematol 2002;30:503–512.PubMedCrossRefGoogle Scholar
  12. 12.
    Zhu G, Chang Y, Zuo J et al. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH. Biochem Biophys Res Commun 2001;281:951–956.PubMedCrossRefGoogle Scholar
  13. 13.
    Goidin D, Mamessier A, Staquet MJ et al. Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopula-tions. Anal Biochem 2001;295:17–21.PubMedCrossRefGoogle Scholar
  14. 14.
    Kammula US, Marincola FM, Rosenberg SA et al. Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination. J Natl Cancer Inst 2000;92:1336–1344.PubMedCrossRefGoogle Scholar
  15. 15.
    Seeger K, Kreuzer KA, Lass U et al. Molecular quantification of response to therapy and remission status in TEL-AML1-positive childhood ALL by real-time reverse transcription polymerase chain reaction. Cancer Res 2001;61:2517–2522.PubMedGoogle Scholar
  16. 16.
    Selvey S, Thompson EW, Matthaei K et al. Beta-actin—an unsuitable internal control for RT-PCR. Mol Cell Probes 2001;15:307–311.PubMedCrossRefGoogle Scholar
  17. 17.
    Raff T, van der Giet M, Endemann D et al. Design and testing of beta-actin primers for RT-PCR that do not co amplify processed pseudogenes. Biotechniques 1997;23:456–460.PubMedGoogle Scholar
  18. 18.
    Solanas M, Moral R, Escrich E. Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors. Anal Biochem 2001;288:99–102.PubMedCrossRefGoogle Scholar
  19. 19.
    Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000;25:169–193.PubMedCrossRefGoogle Scholar
  20. 20.
    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:RE-SEARCH0034.Google Scholar
  21. 21.
    Deng G, Lu Y, Zlotnikov G, Thor AD et al. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 1996;274:2057–2059.PubMedCrossRefGoogle Scholar
  22. 22.
    Bustin SA, Li SR, Phillips S et al. Expression of HLA class II in colorectal cancer: Evidence for enhanced immunogenicity of microsatellite-instability-positive tumours. Tumour Biol 2001;22:294–298.PubMedCrossRefGoogle Scholar
  23. 23.
    Elkahloun AG, Gaudet J, Robinson GS et al. In situ gene expression analysis of cancer using laser capture microdissection, microarrays and real time quantitative PCR. Cancer Biol Ther 2002;1:354–358.PubMedGoogle Scholar
  24. 24.
    Emmert-Buck MR et al. Laser capture microdissection. Science 1996;274:998–1001.PubMedCrossRefGoogle Scholar
  25. 25.
    Walch A, Specht K, Smida J et al. Tissue microdissection techniques in quantitative genome and gene expression analyses. Histochem Cell Biol 2001;115:269–276.PubMedGoogle Scholar
  26. 26.
    Wang E, Miller LD, Ohnmacht GA et al. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 2000;18:457–459.PubMedCrossRefGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2007

Authors and Affiliations

  1. 1.Department of Oncological and Surgical SciencesUniversity of PadovaPadovaItaly

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