Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Open access

Summary

Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute prerequisite for correct normalization and, therefore, for providing accurate and reliable data by quantitative real-time RT PCR gene expression analyses.

1. Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ 2005; 29: 151-9.

2. Pang J, Modlin J, Yolken R. Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol Cell Probes 1992; 6: 251-6.

3. Gut M, Leutenegger CM, Huder JB, Pedersen NC, Lutz H. One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses. J Virol Methods 1999; 77: 37-46.

4. Leutenegger CM, Klein D, Hofmann-Lehmann R, Mislin C, Hummel U, Boni J, et al. Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real-time detection system. J Virol Methods 1999; 78: 105-16.

5. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simul - taneous amplification and detection of specific DNA sequences. Biotechnology (N Y) 1992; 10: 413-17.

6. Morrison TB, Weis JJ, Wittwer CT. Quantification of lowcopy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 1998; 24: 954-8, 960, 962.

7. Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 2004; 32: e103. PMCID: 484200.

8. Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154-60.

9. Clegg RM. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 1992; 211: 353-88.

10. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995; 4: 357-62.

11. Lee LG, Connell CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993; 21: 3761-6. PMCID: 309885.

12. Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5’ nuclease assays. Curr Opin Biotechnol 1998; 9: 43-8.

13. Didenko VV. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Bio - techniques 2001; 31: 1106-16, 18, 1120-1. PMCID: 1941713.

14. Wittwer CT, Herrmann MG, Gundry CN, Elenitoba- Johnson KS. Real-time multiplex PCR assays. Methods 2001; 25: 430-42.

15. Wang T, Brown MJ. mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal Biochem 1999; 269: 198-201.

16. Marras SA, Kramer FR, Tyagi S. Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal 1999; 14: 151-6.

17. Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995-1001.

18. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986-94.

19. Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 1996; 14: 303-8.

20. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 1997; 22: 130-1, 4-8.

21. Whitcombe D, Theaker J, Guy SP, Brown T, Little S. Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 1999; 17: 804-7.

22. 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: 3752-61. PMCID: 110766.

23. Nazarenko I. Homogeneous detection of nucleic acids using self-quenched polymerase chain reaction primers labeled with a single fluorophore (LUX primers). Methods Mol Biol 2006; 335: 95-114.

24. Lowe B, Avila HA, Bloom FR, Gleeson M, Kusser W. Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers. Anal Biochem 2003; 315: 95-105.

25. Nazarenko IA, Bhatnagar SK, Hohman RJ. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res 1997; 25: 2516-21. PMCID: 146748.

26. Svanvik N, Stahlberg A, Sehlstedt U, Sjoback R, Kubista M. Detection of PCR products in real time using light-up probes. Anal Biochem 2000; 287: 179-82.

27. Svanvik N, Westman G, Wang D, Kubista M. Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal Biochem 2000; 281: 26-35.

28. Lukhtanov EA, Lokhov SG, Gorn VV, Podyminogin MA, Mahoney W. Novel DNA probes with low background and high hybridization-triggered fluorescence. Nucleic Acids Res 2007; 35: e30. PMCID: 1865069.

29. Li Q, Luan G, Guo Q, Liang J. A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res 2002; 30: E5. PMCID: 99844.

30. Zhang Y, Zhang D, Li W, Chen J, Peng Y, Cao W. A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Res 2003; 31: e123. PMCID: 219491.

31. Buh Gasparic M, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, et al. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem 2010; 396: 2023-9.

32. Klein D. Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 2002; 8: 257-60.

33. Mocellin S, Rossi CR, Pilati P, Nitti D, Marincola FM. Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 2003; 9: 189-95.

34. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004; 15: 155-66. PMCID: 2291693.

35. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002; 29: 23-39.

36. Bustin SA. Absolute quantification of mRNA using realtime reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25: 169-93.

37. Murphy J, Bustin SA. Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? Expert Rev Mol Diagn 2009; 9: 187-97.

38. Almeida A, Paul Thiery J, Magdelenat H, Radvanyi F. Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling. Anal Biochem 2004; 328: 101-8.

39. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 2006; 27: 126-39.

40. Peters IR, Helps CR, Hall EJ, Day MJ. Real-time RT-PCR: considerations for efficient and sensitive assay design. J Immunol Methods 2004; 286: 203-17.

41. Skern R, Frost P, Nilsen F. Relative transcript quantification by quantitative PCR: roughly right or precisely wrong? BMC Mol Biol 2005; 6: 10. PMCID: 1090581.

42. Cikos S, Bukovska A, Koppel J. Relative quantification of mRNA: comparison of methods currently used for realtime PCR data analysis. BMC Mol Biol 2007; 8: 113. PMCID: 2235892.

43. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RTPCR normalisation; strategies and considerations. Genes Immun 2005; 6: 279-84.

44. Erickson HS, Albert PS, Gillespie JW, Wallis BS, Rodriguez-Canales J, Linehan WM, et al. Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples. Lab Invest 2007; 87: 951-62.

45. Vrana SL, Kluttz BW, Vrana KE. Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum. Brain Res Mol Brain Res 1995; 34: 127-34.

46. Sturzenbaum SR, Kille P. Control genes in quantitative molecular biological techniques: the variability of invariance. Comp Biochem Physiol B Biochem Mol Biol 2001; 130: 281-9.

47. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, et al. Housekeeping genes as internal standards: use and limits. J Biotechnol 1999; 75: 291-5.

48. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004; 313: 856-62.

49. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 2005; 344: 141-3.

50. Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions- specific, validation of references. J Exp Bot 2009; 60: 487-93.

51. Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 2000; 46: 69-81.

52. Ohl F, Jung M, Xu C, Stephan C, Rabien A, Burkhardt M, et al. Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization? J Mol Med (Berl) 2005; 83: 1014-24.

53. Filby AL, Tyler CR. Appropriate ‘housekeeping’ genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol Biol 2007; 8: 10. PMCID: 1802086.

54. Elaković I, Nestorov J, Kovačević S, Matić G. Selection of Reference Genes for Normalization of Real-Time PCR Data in Visceral Adipose Tissue of Female Rats on a Fructose-Enriched Diet. Archives of biological sciences 2012; 64: 1247-59.

55. Tanić N, Perović M, Mladenović A, Ruzdijić S, Kanazir S. Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus-evaluation by real time RT-PCR. J Mol Neurosci 2007; 32: 38-46.

56. Zampieri M, Ciccarone F, Guastafierro T, Bacalini MG, Calabrese R, Moreno-Villanueva M, et al. Validation of suitable internal control genes for expression studies in aging. Mech Ageing Dev 2010; 131: 89-95.

57. McCurley AT, Callard GV. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol 2008; 9: 102. PMCID: 2588455.

58. Al-Bader MD, Al-Sarraf HA. Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR. Brain Res Dev Brain Res 2005; 156: 38-45.

59. Verma AS, Shapiro BH. Sex-dependent expression of seven housekeeping genes in rat liver. J Gastroenterol Hepatol 2006; 21: 1004-8.

60. Derks NM, Muller M, Gaszner B, Tilburg-Ouwens DT, Roubos EW, Kozicz LT. Housekeeping genes revisited: different expressions depending on gender, brain area and stressor. Neuroscience 2008; 156: 305-9.

61. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics 2005; 21: 389-95.

62. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034. PMCID: 126239.

63. Aerts JL, Gonzales MI, Topalian SL. Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR. Biotechniques 2004; 36: 84-6, 8, 90-1.

64. Tristan C, Shahani N, Sedlak TW, Sawa A. The diverse functions of GAPDH: views from different subcellular compartments. Cell Signal 2011; 23: 317-23. PMCID: 3084531.

65. Sirover MA. On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase: biochemical mechanisms and regulatory control. Biochim Biophys Acta 2011; 1810: 741-51.

66. Romans P, Firtel RA, Saxe CL, 3rd. Gene-specific expression of the actin multigene family of Dictyostelium discoideum. J Mol Biol 1985; 186: 337-55.

67. Kusakabe T. Ascidian actin genes: developmental regulation of gene expression and molecular evolution. Zoolog Sci 1997; 14: 707-18.

68. Mehta R, Birerdinc A, Hossain N, Afendy A, Chandhoke V, Younossi Z, et al. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose sam ples. BMC Mol Biol 2010; 11: 39. PMCID: 2886049.

69. Facci MR, Auray G, Meurens F, Buchanan R, van Kessel J, Gerdts V. Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells. Vet Immunol Immunopathol 2011; 141: 11-15.

70. Wang J, Dai J, Jung Y, Wei CL, Wang Y, Havens AM, et al. A glycolytic mechanism regulating an angiogenic switch in prostate cancer. Cancer Res 2007; 67: 149-59.

71. Goidin D, Mamessier A, Staquet MJ, Schmitt D, Berthier- Vergnes O. Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 2001; 295: 17-21.

72. Brunner AM, Yakovlev IA, Strauss SH. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 2004; 4: 14. PMCID: 515301.

73. Nicot N, Hausman JF, Hoffmann L, Evers D. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 2005; 56: 2907-14.

74. Bogdanović MD, Dragičević MB, Tanic NT, Todorović SI, Misic DM, Živković ST, et al. Reverse Transcription of 18S rRNA with Poly(dT)(18) and Other Homopolymers. Plant Mol Biol Rep 2013; 31: 55-63.

75. Adiyanti SS, Loho T. Acute Kidney Injury (AKI) biomarker. Acta Med Indones 2012; 44: 246-55.

76. Liabeuf S, Lenglet A, Desjardins L, Neirynck N, Glorieux G, Lemke HD, et al. Plasma beta-2 microglobulin is associated with cardiovascular disease in uremic patients. Kidney Int 2012; 82: 1297-303.

77. Fung ET, Wilson AM, Zhang F, Harris N, Edwards KA, Olin JW, et al. A biomarker panel for peripheral arterial disease. Vasc Med 2008; 13: 217-24. PMCID: 3133945.

78. Nguewa PA, Agorreta J, Blanco D, Lozano MD, Gomez- Roman J, Sanchez BA, et al. Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens. BMC Mol Biol 2008; 9: 103. PMCID: 2612021.

79. Kwon MJ, Oh E, Lee S, Roh MR, Kim SE, Lee Y, et al. Identification of novel reference genes using multiplatform expression data and their validation for quantitative gene expression analysis. PLoS One 2009; 4: e6162. PMCID: 2703796.

80. Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64: 5245-50.

81. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26: 509-15.

82. Neuvians TP, Gashaw I, Sauer CG, von Ostau C, Kliesch S, Bergmann M, et al. Standardization strategy for quantitative PCR in human seminoma and normal testis. J Biotechnol 2005; 117: 163-71.

83. Maurer-Morelli CV, de Vasconcellos JF, Reis-Pinto FC, Rocha Cde S, Domingues RR, Yasuda CL, et al. A comparison between different reference genes for expression studies in human hippocampal tissue. J Neurosci Methods 2012; 208: 44-7.

84. Brkljačić J, Tanić N, Milutinović DV, Elaković I, Jovanović SM, Perišić T, et al. Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD. BMC Mol Biol 2010; 11: 26. PMCID: 2858027.

85. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55: 611-22.

86. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. Primer sequence disclosure: a clarification of the MIQE guidelines. Clin Chem 2011; 57: 919-21.

87. Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, et al. MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 2010; 11: 74. PMCID: 2955025.

88. Bustin SA. Why the need for qPCR publication guidelines? - The case for MIQE. Methods 2010; 50: 217-26.

89. Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT-qPCR - Publishing data that conform to the MIQE guidelines. Methods 2010; 50: S1-5.

90. Derveaux S, Vandesompele J, Hellemans J. How to do successful gene expression analysis using real-time PCR. Methods 2010; 50: 227-30.

91. Majkić-Singh N, Šumarac Z. Quality Indicators of the Pre-Analytical Phase. J Med Biochem 2012; 31: 174-83.

92. Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M. Properties of the reverse transcription reaction in mRNA quantification. Clin Chem 2004; 50: 509-15.

93. Bolufer P, Lo Coco F, Grimwade D, Barragan E, Diverio D, Cassinat B, et al. Variability in the levels of PML-RAR alpha fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols. Haematologica 2001; 86: 570-6.

Journal of Medical Biochemistry

The Journal of Society of Medical Biochemists of Serbia

Journal Information


IMPACT FACTOR 2017: 1.378
5-year IMPACT FACTOR: 0.704



CiteScore 2017: 1.05

SCImago Journal Rank (SJR) 2017: 0.307
Source Normalized Impact per Paper (SNIP) 2017: 0.532

Cited By

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 290 289 16
PDF Downloads 113 113 12