Real-Time Quantitative Polymerase Chain Reaction in Cardiac Transplant Research

  • Leanne E. Felkin
  • Anne B. Taegtmeyer
  • Paul J. R. Barton
Part of the Methods In Molecular Biology™ book series (MIMB, volume 333)


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.

Key Words

Real-time PCR quantitative PCR mRNA quantification TaqMan RiboGreen RNA degradation genotyping single-nucleotide polymorphism (SNP) polymorphism myocardial biopsy 


  1. 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.CrossRefPubMedGoogle Scholar
  2. 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.CrossRefPubMedGoogle Scholar
  3. 3.
    Heid C. A., Stevens J., Livak K. J., and Williams P. M. (1996) Real time quan-titative PCR. Genome Res. 6, 986–994.CrossRefPubMedGoogle Scholar
  4. 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.PubMedGoogle Scholar
  5. 5.
    Bustin S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. 7.
    Ginzinger D. G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512.CrossRefPubMedGoogle Scholar
  8. 8.
    Brand N. J. and Barton P. J. R. (2002) Myocardial molecular biology: an introduction. Heart 87, 284–293.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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. 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.CrossRefPubMedGoogle Scholar
  12. 12.
    SYBR Green PCR Master Mix and RT-PCR. (2004) Applied Biosystems Protocol: Rev C Part 4310251C.Google Scholar
  13. 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.PubMedGoogle Scholar
  14. 14.
    Primer Express Software v1.5 Applications-based primer design software. (2004) Applied Biosystems User’s Manual: Rev D Part 4303014D.Google Scholar
  15. 15.
    TaqMan Gene Expression Assays. (2004) Applied Biosystems Protocol Rev B 4333458B.Google Scholar
  16. 16.
    Assays-by-Design service for gene expression assays. (2004) Applied Biosystems Protocol Part 4334429C.Google Scholar
  17. 17.
    van Hoof A. and Parker R. (2003) Messenger RNA degradation: beginning at the end. Curr. Biol. 12, R285–R287.CrossRefGoogle Scholar
  18. 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.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 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.CrossRefPubMedGoogle Scholar
  21. 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.CrossRefGoogle Scholar
  22. 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.CrossRefGoogle Scholar
  23. 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.CrossRefPubMedGoogle Scholar
  24. 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.CrossRefPubMedGoogle Scholar
  25. 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. 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.CrossRefPubMedGoogle Scholar
  27. 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.CrossRefPubMedGoogle Scholar
  28. 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, 52–5CrossRefPubMedGoogle Scholar
  29. 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.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. 31.
    Pfaffl M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.CrossRefPubMedGoogle Scholar
  32. 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.CrossRefGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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. 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 32 940–949PubMedGoogle Scholar
  38. 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.CrossRefPubMedGoogle Scholar
  39. 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.CrossRefPubMedGoogle Scholar
  40. 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. 41.
    Assays-on-Demand SNP genotyping products. (2004) Applied Biosystems Protocol Rev A Part 4332856A.Google Scholar
  42. 42.
    Assays-by-Design Service for SNP Assays. (2004) Applied Biosystems Protocol Rev C Part 4334431C.Google Scholar
  43. 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.CrossRefPubMedGoogle Scholar
  44. 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.PubMedGoogle Scholar
  45. 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.CrossRefPubMedGoogle Scholar
  46. 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. 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.CrossRefPubMedGoogle Scholar
  48. 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.CrossRefGoogle Scholar
  49. 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, 1015–10CrossRefPubMedGoogle Scholar
  50. 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.CrossRefPubMedGoogle Scholar
  51. 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.CrossRefPubMedGoogle Scholar
  52. 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.CrossRefPubMedGoogle Scholar
  53. 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.CrossRefGoogle Scholar
  54. 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.Google Scholar
  55. 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.CrossRefPubMedGoogle Scholar
  56. 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.CrossRefPubMedGoogle Scholar
  57. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Leanne E. Felkin
    • 1
  • Anne B. Taegtmeyer
    • 1
  • Paul J. R. Barton
    • 1
  1. 1.National Heart and Lung Institute, Imperial College London, Heart Science CentreHarefield HospitalHarefieldEngland

Personalised recommendations