Abstract
Quantitative PCR arrays are the most reliable and accurate tool for analyzing the expression of a focused panel of genes relevant to a pathway or a disease state. PCR arrays allow gene expression analysis with the sensitivity, dynamic range, and specificity of a real-time PCR as well as the multi-gene profiling capability of a microarray. Differences among real-time PCR kits used in PCR arrays are largely restricted to the DNA polymerases and the detection methods used. In this chapter, we provide a step-by-step protocol for the two detection methods most commonly used in PCR arrays, known as SYBR® Green and TaqMan®, which are based on two different approaches to detect PCR products. While SYBR® Green uses a binding dye that intercalates nonspecifically into double-stranded DNA, the TaqMan® approach relies on a fluorogenic oligonucleotide probe that binds only the DNA sequence between the two PCR primers. Therefore, only specific PCR product can generate a fluorescent signal in TaqMan® PCR. Here we also provide a comparison of the SYBR® Green and TaqMan® approaches and highlight their advantages and disadvantages to help the user to choose the best platform.
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References
Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:75–85
Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 11:1026–1030
Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994
Wang T, Brown MJ (1999) mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal Biochem 269:198–201
Malinen E, Kassinen A, Rinttila T et al (2003) Comparison of real-time PCR with SYBR Green I or 5′-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149:269–277
Schneeberger C, Speiser P, Kury F et al (1995) Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. PCR Methods Appl 4:234–238
Cao H, Shockey JM (2012) Comparison of TaqMan® and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues. J Agric Food Chem 60:12296–12303
Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 5′—3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods 25:402–408
Palmer S, Wiegand AP, Maldarelli F et al (2003) New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41:4531–4536
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
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Alvarez, M.L., Doné, S.C. (2014). SYBR® Green and TaqMan® Quantitative PCR Arrays: Expression Profile of Genes Relevant to a Pathway or a Disease State. In: Alvarez, M., Nourbakhsh, M. (eds) RNA Mapping. Methods in Molecular Biology, vol 1182. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1062-5_27
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DOI: https://doi.org/10.1007/978-1-4939-1062-5_27
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1061-8
Online ISBN: 978-1-4939-1062-5
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