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

  • Rocío Liliana Gómez
  • Lorena Noelia Sendín
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Real time Gene expression Plants 

Notes

Acknowledgments

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

References

  1. 1.
    Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994CrossRefGoogle Scholar
  2. 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 32:1869–1877CrossRefGoogle Scholar
  3. 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 234:13–23CrossRefGoogle Scholar
  4. 4.
    Yi C, Hong Y (2019) Estimating the copy number of transgenes in transformed cotton by real-time quantitative PCR. Methods Mol Biol 1902:137–157CrossRefGoogle Scholar
  5. 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 91:736–742CrossRefGoogle Scholar
  6. 6.
    Lin F, Jiang L, Liu Y et al (2014) Genome-wide identification of housekeeping genes in maize. Plant Mol Biol 86:543–554CrossRefGoogle Scholar
  7. 7.
    Davidson RM, Hansey CN, Gowda M et al (2011) Utility of RNA sequencing for analysis of maize reproductive transcriptomes. Plant Genome 4:191–203CrossRefGoogle Scholar
  8. 8.
    Gause WC, Adamovicz J (1994) The use of the PCR to quantitate gene expression. PCR Methods Appl 3:S123–S135CrossRefGoogle Scholar
  9. 9.
    Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Nat Biotechnol 11:1026–1030CrossRefGoogle Scholar
  10. 10.
    Page AF, Minocha SC (2005) Analysis of gene expression in transgenic plants. Methods Mol Biol 286:291–312PubMedGoogle Scholar
  11. 11.
    Foy CA, Parkes HC (2001) Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem 47:990–1000PubMedGoogle Scholar
  12. 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 4:357–362CrossRefGoogle Scholar
  13. 13.
    Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39CrossRefGoogle Scholar
  14. 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 88:7276–7280CrossRefGoogle Scholar
  15. 15.
    Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308CrossRefGoogle Scholar
  16. 16.
    Whitcombe D, Theaker J, Guy SP et al (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 7:804–807CrossRefGoogle Scholar
  17. 17.
    Thelwell N, Millington S, Solinas A et al (2000) Mode of action and application of scorpion primers to mutation detection. Nucleic Acids Res 28:3752–3761CrossRefGoogle Scholar
  18. 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:e103CrossRefGoogle Scholar
  19. 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 3:85CrossRefGoogle Scholar
  20. 20.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  21. 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 87–112Google Scholar
  22. 22.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  23. 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 10:269CrossRefGoogle Scholar
  24. 24.
    Libault M, Thibivilliers S, Bilgin DD et al (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54CrossRefGoogle Scholar
  25. 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 22:179CrossRefGoogle Scholar
  26. 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 63–82Google Scholar
  27. 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, 876CrossRefGoogle Scholar
  28. 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 2:17CrossRefGoogle Scholar
  29. 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:e43CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Rocío Liliana Gómez
    • 1
  • Lorena Noelia Sendín
    • 1
  1. 1.Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), Estación Experimental Agroindustrial Obispo Colombres (EEAOC)—Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)TucumánArgentina

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