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Cereal Genomics pp 51–63Cite as

Relative Expression Analysis of Target Genes by Using Reverse Transcription-Quantitative PCR

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2072))

Abstract

Real-time PCR is a powerful technique used for quantification of defined nucleic acid sequences. Numerous applications of this method have been described including: gene expression studies, diagnosis of pathogens, and detection of genetically modified organisms or mutations. Here, we describe a simple and efficient protocol to determine gene expression in cereals, based on real-time PCR using SYBR® Green dye. This technique provide an inexpensive alternative, since no probes are required, allowing for the quantification of a high number of genes with reduced cost.

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References

  1. Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994

    Article  CAS  Google Scholar 

  2. Galli V, da Silva Messias R, dos Anjos e Silva SD et al (2013) Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains. Plant Cell Rep 32:1869–1877

    Article  CAS  Google Scholar 

  3. Barbau-Piednoir E, Lievens A, Vandermassen E et al (2012) Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready®, LibertyLink®, and CryIAb traits in genetically modified products. Eur Food Res Technol 234:13–23

    Article  CAS  Google Scholar 

  4. Yi C, Hong Y (2019) Estimating the copy number of transgenes in transformed cotton by real-time quantitative PCR. Methods Mol Biol 1902:137–157

    Article  CAS  Google Scholar 

  5. Malvick DK (2007) Impullitti AE (2007) detection and quantification of Phialophora gregata in soybean and soil samples with a quantitative, real-time PCR assay. Plant Dis 91:736–742

    Article  CAS  Google Scholar 

  6. Lin F, Jiang L, Liu Y et al (2014) Genome-wide identification of housekeeping genes in maize. Plant Mol Biol 86:543–554

    Article  CAS  Google Scholar 

  7. Davidson RM, Hansey CN, Gowda M et al (2011) Utility of RNA sequencing for analysis of maize reproductive transcriptomes. Plant Genome 4:191–203

    Article  CAS  Google Scholar 

  8. Gause WC, Adamovicz J (1994) The use of the PCR to quantitate gene expression. PCR Methods Appl 3:S123–S135

    Article  CAS  Google Scholar 

  9. Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Nat Biotechnol 11:1026–1030

    Article  CAS  Google Scholar 

  10. Page AF, Minocha SC (2005) Analysis of gene expression in transgenic plants. Methods Mol Biol 286:291–312

    CAS  PubMed  Google Scholar 

  11. Foy CA, Parkes HC (2001) Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem 47:990–1000

    CAS  PubMed  Google Scholar 

  12. Livak KJ, Flood SJA, Marmaro J et al (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362

    Article  CAS  Google Scholar 

  13. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39

    Article  CAS  Google Scholar 

  14. 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 (ofigonucleotide probe/human immunodeficiency virus). Proc Natl Acad Sci U S A 88:7276–7280

    Article  CAS  Google Scholar 

  15. Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308

    Article  CAS  Google Scholar 

  16. Whitcombe D, Theaker J, Guy SP et al (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 7:804–807

    Article  Google Scholar 

  17. Thelwell N, Millington S, Solinas A et al (2000) Mode of action and application of scorpion primers to mutation detection. Nucleic Acids Res 28:3752–3761

    Article  CAS  Google Scholar 

  18. Zipper H, Brunner H, Bernhagen J et al (2004) Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 32:e103

    Article  Google Scholar 

  19. Tajadini M, Panjehpour M, Javanmard S (2014) Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res 3:85

    Article  Google Scholar 

  20. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  21. Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) A–Z of quantitative PCR. International University Line, La Jolla, CA, pp 87–112

    Google Scholar 

  22. 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

    Article  CAS  Google Scholar 

  23. Murphy SP, Simmons CR, Bass HW (2010) Structure and expression of the maize (Zea mays L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants. BMC Plant Biol 10:269

    Article  CAS  Google Scholar 

  24. Libault M, Thibivilliers S, Bilgin DD et al (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54

    Article  CAS  Google Scholar 

  25. Li Z, Hansen JL, Liu Y et al (2004) Using real-time PCR to determine transgene copy number in wheat. Plant Mol Biol Rep 22:179

    Article  CAS  Google Scholar 

  26. Pfaffl M (2006) Relative quantification. In: Dorak T (ed) Real-time PCR, Quantification strategies in real-time PCR. International University Line, La Jolla, CA, pp 63–82

    Google Scholar 

  27. Li Z, Trick HN (2005) Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch. BioTechniques 38:872. 874, 876

    Article  CAS  Google Scholar 

  28. Mygind T, Birkelund S, Birkebaek NH et al (2002) Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae. BMC Microbiol 2:17

    Article  Google Scholar 

  29. Schmittgen TD, Jiang J, Liu Q et al (2004) A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 32:e43

    Article  Google Scholar 

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Acknowledgments

This project was supported by Estación Experimental Agroindustrial Obispo Colombres (EEAOC) and Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET).

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Authors and Affiliations

  1. Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), Estación Experimental Agroindustrial Obispo Colombres (EEAOC)—Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Tucumán, Argentina

    Rocío Liliana Gómez & Lorena Noelia Sendín

Authors
  1. Rocío Liliana Gómez
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  2. Lorena Noelia Sendín
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Editors and Affiliations

  1. Instituto de Diversidad y Ecología Animal, Consejo Nacional de Investigaciones Científicas y Técnicas (IDEA, CONICET), Universidad Nacional de Córdoba, Córdoba, Argentina

    Luis M. Vaschetto

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Gómez, R.L., Sendín, L.N. (2020). Relative Expression Analysis of Target Genes by Using Reverse Transcription-Quantitative PCR. In: Vaschetto, L. (eds) Cereal Genomics. Methods in Molecular Biology, vol 2072. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9865-4_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9865-4_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9864-7

  • Online ISBN: 978-1-4939-9865-4

  • eBook Packages: Springer Protocols

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