Quantitative Reverse Transcriptase Polymerase Chain Reaction

  • Lyndon M. Gommersall
  • M. Arya
  • Prabhabhai S. Patel
  • H. R. H. Patel

Abstract

Since the first documentation of real-time polymerase chain reaction (PCR),1 it has been used for an increasing and diverse number of applications, including mRNA expression studies, DNA copy number measurements in genomic or viral DNAs,2–7 allelic discrimination assays,8,9 expression analysis of specific splice variants of genes10–13 and gene expression in paraffin-embedded tissues,14,15 and laser captured microdissected cells.13,16–19 Therefore, quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) is now essential in molecular diagnostics to quantitatively assess the level of RNA or DNA in a given specimen. QRT-PCR enables the detection and quantification of very small amounts of DNA, cDNA, or RNA, even down to a single copy. It is based on the detection of fluorescence produced by reporter probes, which varies with reaction cycle number. Only during the exponential phase of the conventional PCR reaction is it possible to extrapolate back in order to determine the quantity of initial template sequence. The “real-time” nature of this technology pertains to the constant monitoring of fluorescence from specially designed reporter probes during each cycle. Due to inhibitors of the polymerase reaction found with the template, reagent limitation or accumulation of pyrophosphate molecules, the PCR reaction eventually ceases to generate template at an exponential rate (i.e., the plateau phase), making the end point quantitation of PCR products unreliable in all but the exponential phase.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 1993;11:1026–1030.PubMedCrossRefGoogle Scholar
  2. 2.
    Kariyazono H, Ohno T, Ihara K, et al. Rapid detection of the 22q1 1.2 deletion with quantitative real-time PCR. Mol Cell Probes 2001; 15:71–73.PubMedCrossRefGoogle Scholar
  3. 3.
    Nigro JM, Takahashi MA, Ginzinger DG, et al. Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative PCR assay. Am J Pathol 2001;4: 1253–1262.Google Scholar
  4. 4.
    Ginzinger DG, Godfrey TE, Nigro J, et al. Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis. Cancer Res 2000;60:5405–5409.PubMedGoogle Scholar
  5. 5.
    Ingham DJ. The study of transgene copy number and organization. Methods Mol Biol 2005;286:273–290.PubMedGoogle Scholar
  6. 6.
    Bai RK, Perng CL, Hsu CH, Wong LJ. Quantitative PCR analysis of mitochondrial DNA content in patients with mitochondrial disease. Ann NYAcad Sci 2004;1011:304-309.Google Scholar
  7. 7.
    Desire N, Dehee A, Schneider V, et al. Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J Clin Microbiol 2001;39:1303.PubMedCrossRefGoogle Scholar
  8. 8.
    Johnson VJ, Yucesoy B, Luster MI. Genotyping of single nucleotide polymorphisms in cytokine genes using real-time PCR allelic discrimination technology. Cytokine 2004;27:135–141.PubMedCrossRefGoogle Scholar
  9. 9.
    Petersen K, Vogel U, Rockenbauer E, et al. Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms. Mol Cell Probes 2004; 18:117–122.PubMedCrossRefGoogle Scholar
  10. 10.
    Elson D, Thurston G, Huang E, et al. Quiescent angiogenesis in transgenic mice expressing constitutively active hypoxiainducible factor-1a. Genes Dev 2001;15:2520.PubMedCrossRefGoogle Scholar
  11. 11.
    Schmittgen TD, Teske S, Vessella RL, True LD, Zakrajsek BA. Expression of prostate specific membrane antigen and three alternatively spliced variants of PSMA in prostate cancer patients. Int J Cancer 2003; 107:323–329.PubMedCrossRefGoogle Scholar
  12. 12.
    Caberlotto L, Hurd YL, Murdock P, et al. Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. Eur J Neurosci 2003;17:1736–1746.PubMedCrossRefGoogle Scholar
  13. 13.
    Sethi N, Palefsky J. Transcriptional profiling of dysplastic lesions in K14-HPV16 transgenic mice using laser microdissection. FASEB J 2004;18:1243–1245.PubMedGoogle Scholar
  14. 14.
    Godfrey TE, Kim SH, Chavira M, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5’ nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn 2000;2:84–91.PubMedGoogle Scholar
  15. 15.
    Andreassen CN, Sorensen FB, Overgaard J, Alsner J. Optimization and validation of methods to assess single nucleotide polymorphisms (SNPs) in archival histological material. Radiother Oncol 2004;72: 351–356.PubMedCrossRefGoogle Scholar
  16. 16.
    Glockner S, Lehmann U, Wilke N, Kleeberger W, Langer F, Kreipe H. Detection of gene amplification in intraductal and infiltrating breast cancer by laser-assisted microdissection and quantitative realtime PCR. Pathobiology 2000;68:173–179.PubMedCrossRefGoogle Scholar
  17. 17.
    Ehrig T, Abdulkadir SA, Dintzis SM, Milbrandt J, Watson MA. Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection. J Mol Diagn 2001;3:22–25.PubMedGoogle Scholar
  18. 18.
    Fink L, Seeger W, Ermert L, et al. Real-time quantitative RTPCR after laser-assisted cell picking. Nat Med 1998;4:1329–1333.PubMedCrossRefGoogle Scholar
  19. 19.
    Shieh DB, Chou WP, Wei YH, Wong TY, Jin YT. Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR. Ann NYAcad Sci 2004;1011:154.Google Scholar
  20. 20.
    Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991;88:7276–7280.PubMedCrossRefGoogle Scholar
  21. 21.
    Lee LG, Connell CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993;21:3761–3766.PubMedCrossRefGoogle Scholar
  22. 22.
    Cardullo RA, Agrawal S, Flores C, Zamecnick PC, Wolf DE. Detection of nucleic acid hybridization by non-radiative fluorescence resonance energy transfer. Proc Natl Acad Sci USA 1988;85:8790.PubMedCrossRefGoogle Scholar
  23. 23.
    Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–994.PubMedCrossRefGoogle Scholar
  24. 24.
    Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996;6:995–1001.PubMedCrossRefGoogle Scholar
  25. 25.
    Dumur CI, Dechsukhum C, Wilkinson DS, Garrett CT, Ware JL, Ferreira-Gonzalez A. Analytical validation of a real-time reverse transcriptionpolymerase chain reaction quantitation of different transcripts of the Wilms’ tumor suppressor gene (WT1). Anal Biochem 2002;309:127–136.PubMedCrossRefGoogle Scholar
  26. 26.
    Jurado J, Prieto-Alamo MJ, Madrid-Risquez J, Pueyo C. Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse. J Biol Chem 2003;278:45546.PubMedCrossRefGoogle Scholar
  27. 27.
    Borg I, Rohde G, Loseke S, et al. Evaluation of a quantitative realtime PCR for the detection of respiratory syncytial virus in pulmonary diseases. Eur Respir J 2003;21:944–951.PubMedCrossRefGoogle Scholar
  28. 28.
    Lin JC, Wang WY, Chen KY, et al. Quantification of plasma EpsteinBarr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004;350:2461–2470.PubMedCrossRefGoogle Scholar
  29. 29.
    Castelain S, Descamps V, Thibault V, et al. TaqMan amplification system with an internal positive control for HCV RNA quantitation. J Clin Virol 2004;31:227–234.PubMedCrossRefGoogle Scholar
  30. 30.
    Gilliland G, Perrin S, Bunn HF. Competitive PCR for quantitation of mRNA. In: PCR Protocols: A Guide to Methods and Applications. Innis, MA, ed. CA, USA: Academic Press, 1990, 60–69.Google Scholar
  31. 31.
    Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. BioTechniques 2000;29:332–337.PubMedGoogle Scholar
  32. 32.
    Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000;25:169–193.PubMedCrossRefGoogle Scholar
  33. 33.
    Rhoads RP, McManaman C, Ingvartsen KL, Boisclair YR. The housekeeping genes GAPDH and cyclophilin are regulated by metabolic state in the liver of dairy cows. J Dairy Sci 2004;87:248.CrossRefGoogle Scholar
  34. 34.
    Steele BK, Meyers C, Ozbun MA. Variable expression of some “housekeeping” genes during human keratinocyte differentiation. Anal Biochem 2002;307:341–347.PubMedCrossRefGoogle Scholar
  35. 35.
    Yperman J, De Visscher G, Holvoet P, Flameng W. β-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves. J Heart Valve Dis 2004;13:848.PubMedGoogle Scholar
  36. 36.
    Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 2004;37:112, 116, 118.PubMedGoogle Scholar
  37. 37.
    BasA, Forsberg G, Hammarstrom S, Hammarstrom ML. Utility of the housekeeping genes 18S rRNA, β-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 2004;59: 566–573.PubMedCrossRefGoogle Scholar
  38. 38.
    Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:0034.I.CrossRefGoogle Scholar
  39. 39.
    Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 1998;24:954–958, 960, 962.PubMedGoogle Scholar
  40. 40.
    Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997;245:154–160.PubMedCrossRefGoogle Scholar
  41. 41.
    Gibellini D, Vitone F, Schiavone P, Ponti C, La Placa M, Re MC. Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique. J Clin Virol 2004;29: 282–289.PubMedCrossRefGoogle Scholar
  42. 42.
    Blaschke V, Reich K, Blaschke S, Zipprich S, Neumann CJ. Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RT-PCR technology. Immunol Methods 2000;246:79–90.CrossRefGoogle Scholar
  43. 43.
    Ramos-Payan R, Aguilar-Medina M, Estrada-Parra S, et al. Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I. Scand J Immunol 2003;57:439–445.PubMedCrossRefGoogle Scholar
  44. 44.
    Nakamura T, Scorilas A, Stephan C, et al. Quantitative analysis of macrophage inhibitory cytokine-1 (MIC-1) gene expression in human prostatic tissues. Br J Cancer 2003;88:1101–1104.PubMedCrossRefGoogle Scholar
  45. 45.
    Gut M, Leutenegger CM, Huder JB, Pedersen NC, Lutz H. One-tube fluorogenic reverse transcriptionpolymerase chain reaction for the quantitation of feline coronaviruses. J Virol Methods 1999;77: 37–46.PubMedCrossRefGoogle Scholar
  46. 46.
    Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 2001;25:386–401.PubMedCrossRefGoogle Scholar
  47. 47.
    Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002;30:503–512.PubMedCrossRefGoogle Scholar
  48. 48.
    van Hoeyveld E, Houtmeyers F, Massonet C, et al. Detection of single nucleotide polymorphisms in the mannosebinding lectin gene using minor groove binder-DNA probes. J Immunol Methods 2004;287: 227–230.PubMedCrossRefGoogle Scholar
  49. 49.
    de Kok JB, Wiegerinck ET, Giesendorf BA, Swinkels DW. Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum Mutat 2002;19:554–559.PubMedCrossRefGoogle Scholar
  50. 50.
    Zeschnigk M, Bohringer S, Price EA, Onadim Z, Masshofer L, Lohmann DR. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res 2004;32:E125.PubMedCrossRefGoogle Scholar
  51. 51.
    Emig M, Saussele S, Wittor H, et al. Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia 1999;13:1825–1832.PubMedCrossRefGoogle Scholar
  52. 52.
    van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 2003;17: 1013–1034.PubMedCrossRefGoogle Scholar
  53. 53.
    Schalasta G, Eggers M, Schmid M, Enders G. Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system. J Clin Virol 2000;19: 175–185.PubMedCrossRefGoogle Scholar
  54. 54.
    Aliyu SH, Aliyu MH, Salihu HM, Parmar S, Jalal H, Curran MD. Rapid detection and quantitation of hepatitis B virus DNA by realtime PCR using a new fluorescent (FRET) detection system. J Clin Virol 2004;30:191–194.PubMedCrossRefGoogle Scholar
  55. 55.
    Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnol 1996;14:303–308.CrossRefGoogle Scholar
  56. 56.
    Smit ML, Giesendorf BA, Vet JA, Trijbels FJ, Blom HJ. Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons. Clin Chem 2001;47;739–744.PubMedGoogle Scholar
  57. 57.
    Abravaya K, Huff J, Marshall R, et al. Molecular beacons as diagnostic tools: technology and applications. Clin Chem Lab Med 2003;41: 468–474.PubMedCrossRefGoogle Scholar
  58. 58.
    Wabuyele MB, Farquar H, Stryjewski W, et al. Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes. J Am Chem Soc 2003; 125:6937–6945.PubMedCrossRefGoogle Scholar
  59. 59.
    Whitcombe D, Theaker J, Guy SP, Brown T, Little S. Detection of PCR products using self-probing amplicons and flourescence. Nature 1999; 17:804.CrossRefGoogle Scholar
  60. 60.
    Hart KW, Williams OM, Thelwell N, et al. Novel method for detection, typing, and quantification of human papillomaviruses in clinical samples. J Clin Microbiol 2001;39:3204–3212.PubMedCrossRefGoogle Scholar
  61. 61.
    Thelwell N, Millington S, Solinas A, Booth J, Brown T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res 2000;28:3752–3761.PubMedCrossRefGoogle Scholar
  62. 62.
    Solinas A, Brown LJ, McKeen C, et al. Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res 2001;29: E96.PubMedCrossRefGoogle Scholar
  63. 63.
    Ugozzoli LA, Hamby K. Four-color multiplex 5′ nuclease assay for the simultaneous detection of the factor V Leiden and the prothrombin G20210A mutations. Mol Cell Probes 2004;18:161–166.PubMedCrossRefGoogle Scholar
  64. 64.
    Vet JA, Majithia AR, Marras SA, et al. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci USA 1999;96:6394–6399.PubMedCrossRefGoogle Scholar
  65. 65.
    Tong AK, Li Z, Jones GS, Russo JJ, Ju J. Combinatorial fluorescence energy transfer tags for multiplex biological assays. Nature Biotechnol 2001;19:756–759.CrossRefGoogle Scholar
  66. 66.
    Tong AK, Ju J. Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides. Nucleic Acids Res 2002;30:E19.PubMedCrossRefGoogle Scholar
  67. 67.
    Rickman D, Bobek MP, Misek DE, et al. Distinctive molecular profiles of high-grade and low-grade gliomas based on oligonucleotide microarray analysis. Cancer Res 2001;65:6885–6891.Google Scholar
  68. 68.
    Miyazato A, Ueno S, Ohmine K, et al. Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 2001;98:422–427.PubMedCrossRefGoogle Scholar
  69. 69.
    Dolken G. Detection of minimal residual disease. Adv Cancer Res 2001;82:133.PubMedCrossRefGoogle Scholar
  70. 70.
    Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998:62:768–775.PubMedCrossRefGoogle Scholar
  71. 71.
    Hu Y, Zheng M, Xu Z, Wang X, Cui H. Quantitative real-time PCR technique for rapid prenatal diagnosis of Down syndrome. Prenat Diagn 2004;24:704–707.PubMedCrossRefGoogle Scholar
  72. 72.
    Costa C, Pissard S, Girodon E, Huot D, Goossens M. A one-step realtime PCR assay for rapid prenatal diagnosis of sickle cell disease and detection of maternal contamination. Mol Diagn 2003;7:45–48.PubMedCrossRefGoogle Scholar
  73. 73.
    Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 2005;5:209–219.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag London Limited 2007

Authors and Affiliations

  • Lyndon M. Gommersall
    • 1
  • M. Arya
    • 2
  • Prabhabhai S. Patel
    • 3
  • H. R. H. Patel
    • 4
    • 5
  1. 1.Institute of UrologyUniversity College HospitalLondonUK
  2. 2.Molecular Uro-Oncology Research Prostate Cancer Research CentreUniversity College LondonLondonUK
  3. 3.Basic Science ResearchGujarat Cancer and Research Institute Civil HospitalAmedabadIndia
  4. 4.Section of Laparoscopic UrologyUniversity College HospitalLondonUK
  5. 5.Medical CenterUniversity of RochesterRochesterUSA

Personalised recommendations