Skip to main content
SpringerLink
Log in
Book cover

Transplantation Immunology pp 305–330Cite as

Real-Time Quantitative Polymerase Chain Reaction in Cardiac Transplant Research

  • Protocol

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 333))

Abstract

The real-time quantitative polymerase chain reaction (PCR), an increasingly popular technique for the detection of DNA, combines a high degree of accuracy with extreme sensitivity. In this chapter we describe the use of real-time quantitative PCR in trans-plantation research in two areas in which this method is commonly applied: the accurate quantification of mRNA in tissue samples and genotyping of DNA. These are described in the context of cardiac transplantation, but they are of equal relevance to other areas of transplant biology.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Holland P. M., Abramson R. D., Watson R., and Gelfand D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ nuclease actively of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.

    Article  CAS  PubMed  Google Scholar 

  2. Gibson U. E. M., Heid C. A., and Williams P. M. (1996) A novel method for real time quantitative RT-PCR. Genome Res. 6, 995–1001.

    Article  CAS  PubMed  Google Scholar 

  3. Heid C. A., Stevens J., Livak K. J., and Williams P. M. (1996) Real time quan-titative PCR. Genome Res. 6, 986–994.

    Article  CAS  PubMed  Google Scholar 

  4. Livak K. J., Flood S. J., Marmaro J., Giusti W., and Deetz K. (1995) Oligo-nucleotides with fluorescent dyes at opposite ends provide a quenched probe sys-tem useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4, 357–362.

    CAS  PubMed  Google Scholar 

  5. Bustin S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Ginzinger D. G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512.

    Article  CAS  PubMed  Google Scholar 

  8. Brand N. J. and Barton P. J. R. (2002) Myocardial molecular biology: an introduction. Heart 87, 284–293.

    Article  CAS  PubMed  Google Scholar 

  9. Barton P. J. R., Birks E. J., Felkin L. E., Cullen M. E., Koban M. U., and Yacoub M. H. (2003) Increased expression of extracellular matrix regulators TIMP1 and MMP1 in deteriorating heart failure. J. Heart Lung Transplant. 22, 738–744.

    Article  PubMed  Google Scholar 

  10. De Souza A. I., Felkin L. E., Barton P. J. R., Banner N. R., and Rose M. L. (2003) Sequential expression of three known protective genes in cardiac biopsies after transplantation. J. Heart Lung Transplant. 22(1), S163.

    Google Scholar 

  11. Barton P. J. R., Felkin L. E., Koban M. U., Cullen M. E., Brand N. J., and Dhoot G. K. (2004) The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart. Mol. Cell. Biochem. 263, 81–90.

    Article  PubMed  Google Scholar 

  12. SYBR Green PCR Master Mix and RT-PCR. (2004) Applied Biosystems Protocol: Rev C Part 4310251C.

    Google Scholar 

  13. Morrison T. B., Weis J. J., and Wittwer C. T. (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 24, 954–958, 960, 962.

    CAS  PubMed  Google Scholar 

  14. Primer Express Software v1.5 Applications-based primer design software. (2004) Applied Biosystems User’s Manual: Rev D Part 4303014D.

    Google Scholar 

  15. TaqMan Gene Expression Assays. (2004) Applied Biosystems Protocol Rev B 4333458B.

    Google Scholar 

  16. Assays-by-Design service for gene expression assays. (2004) Applied Biosystems Protocol Part 4334429C.

    Google Scholar 

  17. van Hoof A. and Parker R. (2003) Messenger RNA degradation: beginning at the end. Curr. Biol. 12, R285–R287.

    Article  Google Scholar 

  18. Tong D., Schneeberger C., Leodolter S., and Zeillinger R. (1997) Quantitative determination of gene expression by competitive reverse transcription-polymerase chain reaction in degraded RNA samples. Anal. Biochem. 251, 173–177.

    Article  CAS  PubMed  Google Scholar 

  19. Jones L. J., Yue S. T., Cheung C. Y., and Singer V. L. (1998) RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Anal. Biochem. 265, 368–374.

    Article  CAS  PubMed  Google Scholar 

  20. Stahlberg A., Hakansson J., Xian X., Semb H., and Kubista M. (2004) Proper-ties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50(3), 509–515.

    Article  CAS  PubMed  Google Scholar 

  21. Deprez R. H. L., Fijnvandraat A. C., Ruijter J. M., and Moorman A. F. M. (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal. Biochem. 307, 63–69.

    Article  Google Scholar 

  22. Pfaffl M. W. and Hageleit M. (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol. Lett. 23, 275–282.

    Article  CAS  Google Scholar 

  23. Depre C., Shipley G. L., Chen W., et al. (1998) Unloaded heart in vivo repli-cates fetal gene expression of cardiac hypertrophy. Nat. Med. 4, 1269–1275.

    Article  CAS  PubMed  Google Scholar 

  24. Uray I. P., Connelly J. H., Thomazy V., et al. (2002) Left ventricular unloading alters receptor tyrosine kinase expression in the failing human heart. J. Heart Lung Transplant. 21, 771–782.

    Article  PubMed  Google Scholar 

  25. Relative quantitation of gene expression: ABI PRISM 7700 Sequence detection system. (1997) Applied Biosystems User Bulletin #2: Rev B Part 4304859B, pp. 1–36.

    Google Scholar 

  26. Peirson S. N., Butler J. N., and Foster R. G. (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31, e73.

    Article  PubMed  Google Scholar 

  27. Ramakers C., Ruijter J. M., Deprez R. H. L., and Moorman A. F. M. (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62–66.

    Article  CAS  PubMed  Google Scholar 

  28. Liu W. and Saint D. A. (2002) A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302, 52–5

    Article  CAS  PubMed  Google Scholar 

  29. Liu W. and Saint D. A. (2002) Validation of a quantitative method for real time PCR kinetics. Biochem. Biophys. Res. Commun. 294, 347–353.

    Article  CAS  PubMed  Google Scholar 

  30. Pfaffl M. W., Horgan G. W., and Dempfle L. (2002) Relative expression soft-ware tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, e36.

    Article  PubMed  Google Scholar 

  31. Pfaffl M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.

    Article  CAS  PubMed  Google Scholar 

  32. 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, 1–2.

    Article  Google Scholar 

  33. Pfaffl M. W., Tichopad A., Prgomet C., and Neuvians T. P. (2004) Determina-tion of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515.

    Article  CAS  PubMed  Google Scholar 

  34. Livak K. J. and Schmittgen T. D. (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 

  35. Razeghi P., Young M. E., Cockrill T. C., Frazier O. H., and Taegtmeyer H. (2002) Downregulation of myocardial myocyte enhancer factor 2C and myocyte enhancer factor 2C-regulated gene expression in diabetic patients with nonis-chemic heart failure. Circulation 106, 407–411.

    Article  CAS  PubMed  Google Scholar 

  36. Multiple PCR with TaqMan VIC probes: ABI PRISM 7700 sequence detection system. Applied Biosystems User Bulletin 5: Rev B Part 4306236B, 2004.

    Google Scholar 

  37. Afonina I. A. Reed M. W. Lusby E. Shishkina I. G. and Belousov Y. S. 2002 Mir groove binder-conjugated DNA probes for quantitative DNA detec-tion by hybridization-triggered fluorescence. BioTechniques 32 940–949

    CAS  PubMed  Google Scholar 

  38. Afonina I., Zivarts M., Kutyavin I., Lukhtanov E., Gamper H., and Meyer R. B. (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660.

    Article  CAS  PubMed  Google Scholar 

  39. Kutyavin I. V., Lukhtanov E. A., Gamper H. B., and Meyer R. B. (1997) Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization. Nucleic Acids Res. 25, 3718–3723.

    Article  CAS  PubMed  Google Scholar 

  40. Primer express v 1.5 and TaqMan MGB probes for allelic discrimination: All PCR instruments. (2000) Applied Biosystems User Bulletin Part 4317594A, pp. 1–28.

    Google Scholar 

  41. Assays-on-Demand SNP genotyping products. (2004) Applied Biosystems Protocol Rev A Part 4332856A.

    Google Scholar 

  42. Assays-by-Design Service for SNP Assays. (2004) Applied Biosystems Protocol Rev C Part 4334431C.

    Google Scholar 

  43. Cunningham D. A., Crisp S. J., Barbir M., Lazem F., Dunn M. J., and Yacoub M. H. (1998) Donor ACE gene polymorphism: A genetic risk factor for accelerated coronary sclerosis following cardiac transplantation. Eur. Heart J. 19, 319–325.

    Article  CAS  PubMed  Google Scholar 

  44. Loh E., Rebbeck T. R., Mahoney P. D., Denofrio D., Swain J. L., and Holmes E. W. (1999) Common variant in AMPD1 gene predicts improved clinical outcome in patients with heart failure. Circulation 99, 1422–1425.

    CAS  PubMed  Google Scholar 

  45. Anderson J. L., Habashi J., Carlquist J. F., et al. (2000) A common variant of the AMPD1 gene predicts improved cardiovascular survival in patients with coronary artery disease. J. Am. Coll. Cardiol. 36, 1248–1252.

    Article  CAS  PubMed  Google Scholar 

  46. Taegtmeyer A. B., Breen J. B., Smith J. D., et al. (2004) Increased incidence of acute rejection among cardiac transplant recipients possessing the Gln12STOP variant of AMPD-1. Am. J. Transplant. 4(8), 311.

    Google Scholar 

  47. Kalsi K. K., Yuen A. H., Rybakowska I. M., et al. (2003) Decreased cardiac activity of AMP deaminase in subjects with the AMPD1 mutation-A potential mechanism of protection in heart failure. Cardiovasc. Res. 59, 678–684.

    Article  CAS  PubMed  Google Scholar 

  48. Taegtmeyer A. B., Breen J., Smith J. D., Banner N. R., Yacoub M. H., and Barton P. J. (2004) Increased frequency of adenosine monophosphate deaminase 1 C34TT allele in cardiac donors is associated with reduced predonation inotrope. J. Heart Lung Transplant. 23(2), S89.

    Article  Google Scholar 

  49. Owen V. J., Burton P. B. J., Mullen A. J., Birks E. J., Barton P. J. R., and Yacoub M. H. (2001) Expression of RGS3, RGS4 and Gi alpha 2 in acutely fail-ing donor hearts and end-stage heart failure. Eur. Heart J. 22, 1015–10

    Article  CAS  PubMed  Google Scholar 

  50. Radonic A., Thulke S., Mackay I. M., Landt O., Siegert W., and Nitsche A. (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem. Biophys. Res. Commun. 313, 856–862.

    Article  CAS  PubMed  Google Scholar 

  51. Bijlsma F. J., Bruggink A. H., Hartman M., et al. (2001) No association between IL-10 promoter gene polymorphism and heart failure or rejection following cardiac transplantation. Tissue Antigens 57, 151–153.

    Article  CAS  PubMed  Google Scholar 

  52. Gourley I. S., Denofrio D., Rand W., Desai S., Loh E., and Kamoun M. (2004) The effect of recipient cytokine gene polymorphism on cardiac transplant outcome. Hum. Immunol. 65, 248–254.

    Article  CAS  PubMed  Google Scholar 

  53. Densem C. G., Hutchinson I. V., Yonan N., and Brooks N. H. (2003) Influence of interleukin-10 polymorphism on the development of coronary vasculopathy following cardiac transplantation. Transplant. Immunol. 11, 223–228.

    Article  CAS  Google Scholar 

  54. Vamvakopoulos J. E., Taylor C. J., Green C., et al. (2002) Interleukin 1 and chronic rejection: possible genetic links in human heart allografts. Am. J. Trans-plant. 2, 76–83.

    CAS  Google Scholar 

  55. He J. Q., Gaur L. K., Stempien-Otero A., et al. (2002) Genetic variants of the hemostatic system and development of transplant coronary artery disease. J. Heart Lung Transplant. 21, 629–636.

    Article  PubMed  Google Scholar 

  56. Zheng H., Webber S., Zeevi A., et al. (2003) Tacrolimus dosing in pediatric heart transplant patients is related to CYP3A5 and MDR1 gene polymorphisms. Am. J. Transplant. 3, 477–483.

    Article  CAS  PubMed  Google Scholar 

  57. Gonzalez-Amieva A., Lopez-Miranda J., Marin C., et al. (2003) The apo A-I gene promoter region polymorphism determines the severity of hyperlipidemia after heart transplantation. Clin. Transplant. 17, 56–62.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Heart and Lung Institute, Imperial College London, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, England

    Leanne E. Felkin, Anne B. Taegtmeyer & Paul J. R. Barton

Authors
  1. Leanne E. Felkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anne B. Taegtmeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul J. R. Barton
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. National Heart and Lung Institute, London, UK

    Philip Hornick

  2. National Heart and Lung Institute, Harefield, UK

    Marlene Rose

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Felkin, L.E., Taegtmeyer, A.B., Barton, P.J.R. (2006). Real-Time Quantitative Polymerase Chain Reaction in Cardiac Transplant Research. In: Hornick, P., Rose, M. (eds) Transplantation Immunology. Methods In Molecular Biology™, vol 333. Humana Press. https://doi.org/10.1385/1-59745-049-9:305

Download citation

  • DOI: https://doi.org/10.1385/1-59745-049-9:305

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-544-6

  • Online ISBN: 978-1-59745-049-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation