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Selection of Reliable Reference Genes for RT-qPCR Analysis

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Quantitative Real-Time PCR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1160))


Reference genes have become the method of choice for normalization of qPCR data. It has been demonstrated in many studies that reference gene validation is essential to ensure accurate and reliable results. This chapter describes how a pilot study can be set up to identify the best set of reference genes to be used for normalization of qPCR data. The data from such a pilot study should be analyzed with dedicated algorithms such as geNorm to rank genes according to their stability—a measure for how well they are suited for normalization. geNorm also provides insights into the optimal number of reference genes and the overall quality of the selected set of reference genes. Importantly, these results are always in function of the sample type being studied. Guidelines are provided on the interpretation of the results from geNorm pilot studies as well as for the continued monitoring of reference gene quality in subsequent studies. For screening studies including a large, unbiased set of genes (e.g., complete miRNome) an alternative normalization method can be used: global mean normalization. This chapter also describes how the data from such studies can be used to identify reference genes for subsequent validation studies on smaller sets of selected genes.

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  1. Huggett J, Dheda K, Bustin S et al (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  3. Vandesompele J, Kubista M, Pfaffl MW (2009) Real-time PCR: current technology and applications. In: Logan J, Edwards K, Saunders N (eds) Caister Academic, Norfolk, England, pp 1–27

    Google Scholar 

  4. Hellemans J, Preobrazhenska O, Willaert A et al (2004) Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. Nat Genet 36: 1213–1218

    Article  CAS  PubMed  Google Scholar 

  5. Vermeulen J, De Preter K, Naranjo A et al (2009) Predicting outcomes for children with neuroblastoma using a multigene-expression signature: a retrospective SIOPEN/COG/GPOH study. Lancet Oncol 10:663–671

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Mestdagh P, Van Vlierberghe P, De Weer A et al (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 10:R64

    Article  PubMed Central  PubMed  Google Scholar 

  7. Barbara D, Mestdagh P, Hellemans J et al (2012) miRNA expression profiling: from reference genes to global mean normalization. Methods Mol Biol 822:261–272

    Article  Google Scholar 

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Correspondence to Jan Hellemans .

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Hellemans, J., Vandesompele, J. (2014). Selection of Reliable Reference Genes for RT-qPCR Analysis. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 1160. Humana Press, New York, NY.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0732-8

  • Online ISBN: 978-1-4939-0733-5

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