Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet 1999;21:33–37.
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.
hang L, Zhou W, Velculescu VE et al. Gene expression profiles in normal and cancer cells. Science 1997;276:1268–1272.
Heid CA, Stevens J, Livak KJ et al. Real time quantitative PCR. Genome Res 1996;6:986–994.
Klein D. Quantification using real-time PCR technology: Applications and limitations. Trends Mol Med 2002;8:257–260.
Higuchi R, Dollinger G, Walsh PS et al. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 1992;10:413–417.
Lee LG, Connell CR, Bloch W et al. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993;21:3761–3766.
Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5′ Nuclease assays. Curr Opin Biotechnol 1998;9:43–48.
Didenko W. DNA probes using fluorescence resonance energy transfer (FRET): Designs and applications. Biotechniques 2001;31:1106–1116.
Wittwer CT, Herrmann MG, Gundry CN et al. Real-time multiplex PCR assays. Methods 2001;25:430–442.
Ginzinger DG. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Exp Hematol 2002;30:503–512.
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.
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.
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.
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.
Selvey S, Thompson EW, Matthaei K et al. Beta-actin—an unsuitable internal control for RT-PCR. Mol Cell Probes 2001;15:307–311.
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.
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.
Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000;25:169–193.
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.
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.
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.
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.
Emmert-Buck MR et al. Laser capture microdissection. Science 1996;274:998–1001.
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.
Wang E, Miller LD, Ohnmacht GA et al. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 2000;18:457–459.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Landes Bioscience and Springer Science+Business Media
About this chapter
Cite this chapter
Provenzano, M., Mocellin, S. (2007). Complementary Techniques. In: Mocellin, S. (eds) Microarray Technology and Cancer Gene Profiling. Advances in Experimental Medicine and Biology, vol 593. Springer, New York, NY. https://doi.org/10.1007/978-0-387-39978-2_7
Download citation
DOI: https://doi.org/10.1007/978-0-387-39978-2_7
Publisher Name: Springer, New York, NY
Print ISBN: 978-0-387-39977-5
Online ISBN: 978-0-387-39978-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)