Current approaches to development of real-time qPCR test-kits

Currently fluorescent quantitative real-time polymerase chain reaction, which is a cutting-edge technology in genetic diagnosis, is used in different areas of molecularbiology. Practicaladvantageofsimplicityaswellascombination of high speed, sensitivityandspecificitymadeitpossibletousethisanalysisfor nucleic acidquantitation. Thepaperpresentsgeneral information andrecommendedrules for thedevelopmentof real-timeqPCR. Thepublication isaimedtoacquaint theresearchersandreviewerswith necessaryrequirementstobefollowedin order toensure high accuracy, reliabilityandtransparencyof theexperiments, correct interpretation andrepeatabilityof thetest results. Currentapproachesaredescribedthatallow obtainingreliableandconsistent resultsbydifferentoperators, at differenttimesandin differentlaboratories. Basicrequirementsforreagentsused, nucleotidesequencesandvalidationmethodsaregiven. In general, thepublication givestheinformation neededtoachievethreeultimategoals: toprovidetheauthorswith abroadrangeoftoolsandrequirementsforthedevelopmentof real-time qPCR based-techniques; togivethepossibilitytothereviewersandeditorsofassessingthequalityofarticlesandguidelines/instructions in accordance with the requiredcriteria; toobtain consistentandreliableresultsof testsperformedusingthis method. 

1. Bernard P. S., Wittwer C. T. Real-time PCR technology for cancer diagnostics. Clin. Chem. 2002; 48 (8): 1178–1185. PMID: 12142370.

2. Burns M. J., Valdivia H., Harris N. Analysis and interpretation of data from real-time PCR trace detection methods using quantitation of GM soya as a model system. Anal. Bioanal. Chem. 2004; 378 (6): 1616–1623. DOI: 10.1007/s00216-003-2441-9.

3. Bustin S. A., Benes V., Nolan T., Pfaffl M. W. Quantitative real-time RTPCR – a perspective. J. Mol. Endocrinol. 2005; 34 (3): 597–601. DOI: 10.1677/jme.1.01755.

4. Bustin S. A., Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin. Sci. (Lond.). 2005; 109 (4): 365–379. DOI: 10.1042/CS20050086.

5. Bustin S. A., Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 2004; 15 (3): 155–166. PMID: 15331581.

6. Damond F., Benard A., Ruelle J., Alabi A., Kupfer B., Gomes P., et al. Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006. J. Clin. Microbiol. 2008; 46 (6): 2088–2091. DOI: 10.1128/JCM.00126-08.

7. Bustin S. A., Mueller R. Real-time reverse transcription PCR and the detection of occult disease in colorectal cancer. Mol. Aspects Med. 2006; 27 (2–3): 192–223. DOI: 10.1016/j.mam.2005.12.002.

8. Ferns R. B., Garson J. A. Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control. J. Virol. Methods. 2006; 135 (1): 102–108. DOI: 10.1016/j.jviromet.2006.02.005.

9. Garson J. A., Grant P. R., Ayliffe U., Ferns R. B., Tedder R. S. Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirusinternal control. J. Virol. Methods. 2005; 126 (1–2): 207–213. DOI: 10.1016/j.jviromet.2005.03.001.

10. Garson J. A., Huggett J. F., Bustin S. A., Pfaffl M. W., Benes V., Vandesompele J., Shipley G. L. Unreliable real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA expression and DNA copy number in multiple sclerosis. AIDS Res. Hum. Retroviruses. 2009; 25 (3): 377–378. DOI: 10.1089/aid.2008.0270.

11. HuangT., Li L. T., Bernstam E. V., Jiang X. Confidence-based laboratory test reduction recommendation algorithm. BMC Med. Inform. Decis. Mak. 2023; 23 (1):93. DOI: 10.1186/s12911-023-02187-3.

12. Shaw A., Reid S. M., Ebert K., Hutchings G., Ferris N., King D. Implementation of a one-step real-time RT-PCR protocol for diagnosis of footand-mouth disease. J. Virol. Methods. 2007; 143 (1): 81–85. DOI: 10.1016/j.jviromet.2007.02.009.

13. Schmidt M. W., Houseman A., Ivanov A. R., Wolf D. A. Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe. Mol. Syst. Biol. 2007; 3:79. DOI: 10.1038/msb4100117.

14. Wittwer C. T., Herrmann M. G., Moss A. A., Rasmussen R. P. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques. 1997; 22 (1): 130–138. DOI: 10.2144/97221bi01.

15. Wittwer С. T., Kusakawa N. Real-time PСR. In: MoleсularMiсrobiology: Diagnostiс Prinсiples and Praсtiсe. Ed. by D. H. Persing, F. С. Tenover, J. Versaloviс, J. W. Tang, E. R. Unger, D. A. Relman, T. J. White. Washington: ASM Press; 2004; 71–84.

16. Bjornsson H. T., Albert T. J., Ladd-Acosta C. M., Green R. D., Rongione M. A., Middle C. M., et al. SNP-specific array-based allele-specific expression analysis. Genome Res. 2008; 18 (5): 771–779. DOI: 10.1101/gr.073254.107.

17. Brazma A., Hingamp P., Quaсkenbush J., Sherloсk G., Spellman P., Stoeсkert С., et al. Minimum information about a miсroarray experiment (MIAME) – toward standards for miсroarray data. Nat. Genet. 2001; 29 (4): 365–371. DOI: 10.1038/ng1201-365.

18. Burns M., Valdivia H. Modelling the limit of deteсtion in real-time quantitative PСR. Eur. FoodRes. Teсhnol. 2008; 226: 1513–1524. DOI: 10.1007/s00217-007-0683-z.

19. Bustin S. A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 2000; 25 (2): 169–193. DOI: 10.1677/jme.0.0250169.

20. Doma M. K., Parker R. RNA quality control in eukaryotes. Cell. 2007; 131 (4): 660–668. DOI: 10.1016/j.cell.2007.10.041.

21. Fleige S., Pfaffl M. W. RNA integrity and the effect on the realtime qRT-PCR performance. Mol. Aspects Med. 2006; 27 (2–3): 126–139. DOI: 10.1016/j.mam.2005.12.003.

22. Burns M. J., Nixon G. J., Foy C. A., Harris N. Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves. BMCBiotechnol. 2005; 5:31. DOI: 10.1186/1472-6750-5-31.

23. Bustin S. A. Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Rev. Mol. Diagn. 2005; 5 (4): 493–498. DOI: 10.1586/14737159.5.4.493.

24. Gingeras T. R. RNA reference materials for gene expression studies. Difficult first steps. Clin. Chem. 2004; 50 (8): 1289–1290. DOI: 10.1373/clinchem.2003.030072.

25. Hellemans J., Mortier G., De Paepe A., Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007; 8 (2):R19. DOI: 10.1186/gb-2007-8-2-r19.

26. Karlen Y., MсNair A., Perseguers S., Mazza С., Mermod N. Statistiсal signifiсanсe of quantitative PСR. BMС Bioinformatiсs. 2007; 8:131. DOI: 10.1186/1471-2105-8-131.

27. Lefever S., Hellemans J., Pattyn F., Przybylski D. R., Taylor C., Geurts R., et al. RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009; 37 (7): 2065–2069. DOI: 10.1093/nar/gkp056.

28. Ellison S. L., English C. A., Burns M. J., Keer J. T. Routes to improving the reliability of low levelDNA analysis using real-time PCR. BMC Biotechnol. 2006; 6:33. DOI: 10.1186/1472-6750-6-33.

29. Huggett J., Dheda K., Bustin S., Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005; 6 (4): 279–284. DOI: 10.1038/sj.gene.6364190.

30. Micke P., Ohshima M., Tahmasebpoor S., Ren Z. P., OstmanA., Pontén F., Botling J. Biobanking of fresh frozen tissue: RNAisstable in nonfixed surgical specimens. Lab. Invest. 2006; 86 (2): 202–211. DOI: 10.1038/labinvest.3700372.

31. Kubista M., Andrade J. M., Bengtsson M., Forootan A., Jonák J., Lind K., et al. The real-time polymerase chain reaction. Mol. Aspects Med. 2006; 27 (2–3): 95–125. DOI: 10.1016/j.mam.2005.12.007.

32. Böhlenius H., Eriksson S., Parсy F., Nilsson O. Retraсtion. Sсienсe. 2007; 316:367. DOI: 10.1126/science.316.5823.367b.

33. Echeverri C. J., Beachy P. A., Baum B., Boutros M., Buchholz F., Chanda S. K., et al. Minimizingtheriskofreportingfalsepositivesinlarge-scaleRNAi screens. Nat. Methods. 2006; 3 (10): 777–779. DOI: 10.1038/nmeth1006-777.

34. Landi D., Gemignani F., Naccarati A., Pardini B., Vodicka P., Vodickova L., et al. Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer. Carcinogenesis. 2008; 29 (3): 579–584. DOI: 10.1093/carcin/bgm304.

35. Mackay I. M., Arden K. E., Nitsche A. Real-time PCR in virology. Nucleic Acids Res. 2002; 30 (6): 1292–1305. DOI: 10.1093/nar/30.6.1292.

36. Mackay I. M. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 2004; 10 (3): 190–212. DOI: 10.1111/j.1198-743x.2004.00722.x.

37. Morrogh M., Olvera N., Bogomolniy F., Borgen P. I., King T. A. Tissue preparation for laser capture microdissection and RNA extraction from fresh frozen breast tissue. BioTechniques. 2007; 43 (1): 41–42, 44, 46. DOI: 10.2144/000112497.

38. Nolan T., Hands R. E., Ogunkolade W., Bustin S. A. SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal. Biochem. 2006; 351 (2): 308–310. DOI: 10.1016/j.ab.2006.01.051.

39. Taylor C. F., Field D., Sansone S. A., Aerts J., Apweiler R., Ashburner M., et al. Promoting coherent minimum reporting guidelinesfor biological and biomedical investigations: the MIBBI project. Nat. Biotechnol. 2008; 26 (8): 889–896. DOI: 10.1038/nbt.1411.

40. Joseph L. J. RNA reference materials for gene expression studies. RNA metrology: forecast calls for partial clearing. Clin. Chem. 2004; 50 (8): 1290–1292. DOI: 10.1373/clinchem.2004.032441.

41. Cronin M., Ghosh K., Sistare F., Quackenbush J., VilkerV., O’Connell C. Universal RNA reference materials for gene expression. Clin. Chem. 2004; 50 (8): 1464–1471. DOI: 10.1373/clinchem.2004.035675.

42. MSI Board Members; Sansone S. A., Fan T., Goodacre R., Griffin J. L., Hardy N. W., et al. The metabolomics standards initiative. Nat. Biotechnol. 2007; 25 (8): 846–848. DOI: 10.1038/nbt0807-846b.

43. Gygi S. P., Rochon Y., Franza B. R., Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 1999; 19 (3): 1720– 1730. DOI: 10.1128/MCB.19.3.1720.

44. Reiter M., Pfaffl M. W. Effects of plate position, plate type and sealing systems on real-time PCR results. Biotechnology & Biotechnological Equipment. 2008; 22 (3): 824–828. DOI: 10.1080/13102818.2008.10817561.

45. Ben-Dov C., Hartmann B., Lundgren J., Valcárcel J. Genome-wide analysis of alternative pre-mRNA splicing. J. Biol. Chem. 2008; 283 (3): 1229– 1233. DOI: 10.1074/jbc.R700033200.

46. Higuchi R., Dollinger G., Walsh P. S., Griffith R. Simultaneous amplification and detection of specific DNA sequences. Nat. Biotechnol. 1992; 10 (4): 413–417. DOI: 10.1038/nbt0492-413.

47. Huggett J. F., Novak T., Garson J. A., Green C., Morris-Jones S. D., Miller R. F., Zumla A. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res. Notes. 2008; 1:70. DOI: 10.1186/1756-0500-1-70.

48. Schmittgen T. D., Livak K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008; 3 (6): 1101–1108. DOI: 10.1038/nprot.2008.73.

49. Ståhlberg A., Håkansson J., Xian X., SembH., Kubista M. Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 2004; 50 (3): 509–515. DOI: 10.1373/clinchem.2003.026161.

50. Taylor C. F., Paton N. W., Lilley K. S., Binz P. A., Julian R. K. Jr., Jones A. R., et al. The minimum information about a proteomics experiment (MIAPE). Nat. Biotechnol. 2007; 25 (8): 887–893. DOI: 10.1038/nbt1329.

51. Vogelstein B., Kinzler K. W. Digital PCR. Proc.Natl. Acad. Sci. USA. 1999; 96 (16): 9236–9241. DOI: 10.1073/pnas.96.16.9236.

52. Dube S., Qin J., Ramakrishnan R. Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One. 2008; 3 (8):e2876. DOI: 10.1371/journal.pone.0002876.

53. FieldD., Garrity G., Gray T., Morrison N., SelengutJ., Sterk P., et al. The minimum information about a genome sequence (MIGS) specification. Nat. Biotechnol. 2008; 26 (5): 541–547. DOI: 10.1038/nbt1360.

54. Higuchi R., Fockler C., Dollinger G., Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Nat. Biotechnol. 1993; 11 (9): 1026–1030. DOI: 10.1038/nbt0993-1026.

55. Nolan T., Hands R. E., Bustin S. A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 2006; 1 (3): 1559–1582. DOI: 10.1038/nprot.2006.236.

56. Pattyn F., Speleman F., De Paepe A., Vandesompele J. RTPrimerDB: the real-time PCR primer and probe database. NucleicAcids Res. 2003; 31 (1): 122–123. DOI: 10.1093/nar/gkg011.

57. Pfaffl M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29 (9):e45. DOI: 10.1093/nar/29.9.e45.

58. Ramsden S. C., Daly S., Geilenkeuser W. J., Duncan G., Hermitte F., Marubini E., et al. EQUAL-quant: an international external quality assessment scheme for real-time PCR. Clin. Chem. 2006; 52 (8): 1584–1591. DOI: 10.1373/clinchem.2005.066019.

59. Breslauer K. J., Frank R., Blöcker H., Marky L. A. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA. 1986; 83 (11): 3746–3750. DOI: 10.1073/pnas.83.11.3746.

60. Sugimoto N., Nakano S., Katoh M., Matsumura A., Nakamuta H., Ohmichi T., et al. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry. 1995; 34 (35): 11211–11216. DOI: 10.1021/bi00035a029.

61. Xia T., SantaLucia J. Jr., Burkard M. E., Kierzek R., Schroeder S. J., Jiao X., et al. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry. 1998; 37 (42): 14719–14735. DOI: 10.1021/bi9809425.

62. Elnifro E. M., Ashshi A. M., Cooper R. J., Klapper P. E. Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 2000; 13 (4): 559–570. DOI: 10.1128/CMR.13.4.559.

63. Wittwer C. T., Herrmann M. G., Gundry C. N., Elenitoba-Johnson K. S. Real-time multiplex PCR assays. Methods. 2001; 25 (4): 430–442. DOI: 10.1006/meth.2001.1265.

64. Russia State Pharmacopoeia. Part 1. 12th ed. Moscow: Scientific Centre for Expert Evaluation of Medicinal Products; 2008. 696 p. (in Russ.)

65. Lakin G. F. Biometrics. 4th ed., revised and supplemented. Moscow: Vysshaya shkola; 1990. 351 p. (in Russ.)

66. Носырев П., Носырева М., Рассказова Т., Корнеева Н. Валидация аналитических методик: теория и практика (часть I). Ремедиум. 2003; 10; 69–71. EDN: PLLZDJ.

67. Swango K. L., Hudlow W. R., Timken M. D., Buoncristiani M. R. Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples. Forensic Sci. Int. 2007; 170 (1): 35–45. DOI: 10.1016/j.forsciint.2006.09.002.

68. Principles and methods of validation of diagnostic assays for infectious diseases. In: WOAH. Manual of Diagnostic Tests and Vaccinesfor TerrestrialAnimals. Paris; 2018; Chapter 1.1.6. Режим доступа: https://www.woah. org/fileadmin/Home/eng/Health_standards/tahm/1.01.06_VALIDATION. pdf (дата обращения: 20.01.2013).

69. OIE. Quality Standard and Guidelines for Veterinary Laboratories: Infeсtious Diseases. 2nd ed. Paris; 2008. 70 p.

70. GOST R ISO 5725-1-2002 Accuracy (trueness and precision) of measurement methods and results. Part 1–6. Available at: https://docs.cntd.ru/document/1200029975. (in Russ.)

71. Haney S. A. Increasing the robustness and validity of RNAi screens. Pharmacogenomics. 2007; 8 (8): 1037–1049. DOI: 10.2217/14622416.8.8.1037.

72. VandesompeleJ., DePreterK., PattynF., PoppeB., VanRoyN., DePaepeA., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3 (7): research0034.1–0034.11. DOI: 10.1186/gb-2002-3-7-research0034.

73. Andersen C. L., Jensen J. L., Ørntoft T. F. 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 (15): 5245–5250. DOI: 10.1158/0008-5472.CAN-04-0496.