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Quantitative PCR for Detection of mRNA and gDNA in Environmental Isolates

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Environmental Microbiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1096))

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Abstract

Quantitative PCR is used to gauge the abundance of specific nucleic acid species within purified samples. Due to its high sensitivity and minimal operation costs, this method is routinely applied in modern molecular bioscience laboratories. Nonetheless, all quantitative PCR experiments must include several carefully designed, yet simple, controls to ensure the reliability of the analyses. The aim of this chapter is to provide basic quantitative PCR methods, from primer design through data analysis, that are generally applicable to studies in microbiology. These methods allow the abundance of targeted RNA or DNA molecules to be determined in nucleic acid samples purified from a variety of biological sources.

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References

  1. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  2. VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626

    Article  CAS  PubMed  Google Scholar 

  3. Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030

    Article  CAS  PubMed  Google Scholar 

  4. Holland PM, Abramson RD, Watson R, Gelfand DH (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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    CAS  PubMed  Google Scholar 

  7. You FM, Huo N, Gu YQ, Luo MC, Ma Y, Hane D, Lazo GR, Dvorak J, Anderson OD (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253

    Article  PubMed Central  PubMed  Google Scholar 

  8. Brzoska AJ, Hassan KA, de Leon EJ, Paulsen IT, Lewis PJ (2013) Single-step selection of drug resistant Acinetobacter baylyi ADP1 mutants reveals a functional redundancy in the recruitment of multidrug efflux systems. PLoS ONE 8:e56090

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  10. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  13. Hassan KA, Brzoska AJ, Wilson NL, Eijkelkamp BA, Brown MH, Paulsen IT (2011) Roles of DHA2 family transporters in drug resistance and iron homeostasis in Acinetobacter spp. J Mol Microbiol Biotechnol 20:116–124

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Part of this work was supported by Project Grant 535053 from the National Health and Medical Research Council. KH is supported by an Australian Postdoctoral Fellowship funded by the Australian Research Council (grant DP110102680).

Author information

Authors and Affiliations

  1. School of Biological Sciences, University of Sydney, Sydney, NSW, Australia

    Anthony J. Brzoska

  2. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

    Karl A. Hassan

Authors
  1. Anthony J. Brzoska
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  2. Karl A. Hassan
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Editor information

Editors and Affiliations

  1. Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia

    Ian T. Paulsen

  2. School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia

    Andrew J. Holmes

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Brzoska, A.J., Hassan, K.A. (2014). Quantitative PCR for Detection of mRNA and gDNA in Environmental Isolates. In: Paulsen, I., Holmes, A. (eds) Environmental Microbiology. Methods in Molecular Biology, vol 1096. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-712-9_3

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  • DOI: https://doi.org/10.1007/978-1-62703-712-9_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-711-2

  • Online ISBN: 978-1-62703-712-9

  • eBook Packages: Springer Protocols

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