Abstract
This chapter describes methods for quantitation of gene expression in bone cells and bone tissues using the technique of quantitative reverse transcription-polymerase chain reaction.
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References
Saiki, R., Scharf, S., Faloona, F., Mullis, K., Hom, G., and Erlich, H. (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression using real-time quantitative PCR and the 2-deltadeltaCT method. Methods 25, 402–408.
Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.
Thellin, O., Zorz,i W., Lakaye, B., De Borman, B., Coumans, B, Hennen, G., Grisar, T., Igout, A., and Heinen, E. (1999) Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75, 291–295.
Dheda, K., Huggett, J. F., Bustin, S. A., Johnson, M. A., Rook, G., and Zumla, A. (2004) Validation of housekeeping genes for nomalizing RNA expression in real-time PCR. Biotechniques 37, 112–119.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 34.
Gilsbach, R., Kouta, M., Bonisch, H., and Bruss, M. (2006) Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data. Biotechniques 40, 173–177.
Nolan, T., Hands, R. E., and Bustin, S. A. (2006) Quantification of mRNA using real-time RT-PCR. Nat. Protocols 1, 1559–1582.
Rozen, S., and Skaletsky, H. J. (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365–386.
Bower, N. I., Moser, R. J., Hill, J. R., and Lehnert, S. A. (2007) Universal reference method for real-time PCR gene expression analysis of preimplantation embryos. Biotechniques 42, 199–206.
Pfaffl, M. W., Hageleit, M. (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotech. Letts. 23, 275–282.
Pfaffl, M. W., Horgan, G. W., and Dempfle, L. (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative gene expression results in real-time PCR. Nucleic Acids Res. 30, e36.
Karlen, Y., McNair, A., Perseguers, S., Mazza, C., and Mermod, N. (2007) Statistical analysis of quantitative PCR. BMC Bioinformatics 8, 131.
Yuan, Y. S., Reed, A., Chen, F., and Stewart, C. N. Jr. (2006) Statistical analysis of real-time PCR data. BMC Bioinformatics 7, 85.
Stahlberg, A., Hakansson, J., Xian, X., Semb, H., and Kubista, M. (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50, 509–515.
Fronhoffs, S., Tozke, G., Stier, S., Wernert, N., Rothe, M., Bruning, T., Koch, B., Sachinidis, A., Vetter, H., and Ko, Y. (2002) A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol. Cell Probes 16, 99–110.
Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.
Whelan, J. A., Russel, N. B., and Whelan, M. A. (2003) A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods 278, 261–269.
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Hughes, A. (2012). Analysis of Gene Expression in Bone by Quantitative RT/PCR. In: Helfrich, M., Ralston, S. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 816. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-415-5_18
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DOI: https://doi.org/10.1007/978-1-61779-415-5_18
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-415-5
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