Abstract
qPCR instruments are supplied with basic software packages that enable the measurement of fluorescent changes, calculations of quantification cycle (C q ) values, the generation of standard curves and subsequent relative target nucleic acid quantity determination. However, detailed assessments of the technical parameters underlying C q values and their translation into biological meaningful results require validation of these basic calculations through further analyses such as qPCR efficiency correction, normalization to multiple reference genes, averaging and statistical tests. Some instruments incorporate some of these features, while others offer additional tools to complement the basic running software, in many cases providing those that are described below. In this chapter, there is a detailed description of some of these programs and recommended strategies for the design of robust qPCR assays. Some of the packages available for validation of the resulting C q data and detailed statistical analysis are described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bustin, S. A., Benes, V., Garson, J. A., et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55, 611–622.
Bustin, S. A. (2010) Why the need for qPCR publication guidelines? – The case for MIQE. Methods 50, 217–226.
Crofts, L. A., Hancock, M. S., Morrison, N. A., and Eisman, J. A. (1998) Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts, Proc Natl Acad Sci USA 95, 10529–10534.
Lefever, S., Vandesompele, J., Speleman, F., and Pattyn, F. (2009) RTPrimerDB: the portal for real-time PCR primers and probes. Nucleic Acids Res 37, D942–D945.
Pattyn, F., Robbrecht, P., De Paepe, A., et al. (2006) RTPrimerDB: the real-time PCR primer and probe database, major update 2006. Nucleic Acids Res 34, D684–D688.
Pattyn, F., Speleman, F., De Paepe, A., and Vandesompele, J. (2003) RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res 31, 122–123.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.
SantaLucia, J., Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95, 1460–1465.
Bommarito, S., Peyret, N., and SantaLucia, J., Jr. (2000) Thermodynamic parameters for DNA sequences with dangling ends. Nucl Acids Res 28, 1929–1934.
Peyret, N., Seneviratne, P. A., Allawi, H. T., and SantaLucia, J., Jr. (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38, 3468–3477.
Allawi, H. T., and SantaLucia, J., Jr. (1998) Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects. Biochemistry 37, 9435–9444.
Allawi, H. T., and SantaLucia, J., Jr. (1998) Thermodynamics of internal C.T mismatches in DNA. Nucleic Acids Res 26, 2694–2701.
Allawi, H. T., and SantaLucia, J., Jr. (1998) Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA. Biochemistry 37, 2170–2179.
Allawi, H. T., and SantaLucia, J., Jr. (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10581–10594.
Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288, 911–940.
He, L., Kierzek, R., SantaLucia, J., Jr., et al. (1991) Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text]. Biochemistry 30, 11124–11132.
SantaLucia, J., Jr., Kierzek, R., and Turner, D. H. (1991) Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs. Biochemistry 30, 8242–8251.
SantaLucia, J., Jr., Kierzek, R., and Turner, D. H. (1990) Effects of GA mismatches on the structure and thermodynamics of RNA internal loops. Biochemistry 29, 8813–8819.
Pfaffl, M. W., Horgan, G. W., and Dempfle, L. (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36.
Andersen, C. L., Jensen, J. L., and Orntoft, T. F. (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.
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, 0034.0031–0034.0011.
Hellemans, J., Mortier, G., De Paepe, A., et al. (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8, R19.
Pabinger, S., Thallinger, G. G., Snajder, R., et al. (2009) QPCR: Application for real-time PCR data management and analysis. BMC Bioinformatics 10, 268.
Lefever, S., Hellemans, J., Pattyn, F., et al. (2009) RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res 37, 2066–2069.
Acknowledgements
S.A.B. would like to thank the charity B&CR (Charity Number 1119105) for support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bustin, S., Bergkvist, A., Nolan, T. (2011). In Silico Tools for qPCR Assay Design and Data Analysis. In: Yu, B., Hinchcliffe, M. (eds) In Silico Tools for Gene Discovery. Methods in Molecular Biology, vol 760. Humana Press. https://doi.org/10.1007/978-1-61779-176-5_18
Download citation
DOI: https://doi.org/10.1007/978-1-61779-176-5_18
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-175-8
Online ISBN: 978-1-61779-176-5
eBook Packages: Springer Protocols