Design of Primers and Probes for Quantitative Real-Time PCR Methods

  • Alicia Rodríguez
  • Mar Rodríguez
  • Juan J. Córdoba
  • María J. AndradeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)


Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR® Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR).

This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

Key words

Quantitative real-time PCR Primers Probes Software and databases Validation Reverse transcription real-time PCR 



We acknowledge financial support of this work by projects “AGL2010-21623” and “Carnisenusa CSD2007-00016—Consolider Ingenio 2010” of the Spanish Government and GR10162 of the Government of Extremadura and FEDER.


  1. 1.
    Invitrogen (2008) Real-time PCR: from theory to practice. Accessed 6 Nov 2013
  2. 2.
    Rodríguez-Lázaro D, Hernández M (2013) Real time PCR in food science: introduction. Curr Issues Mol Biol 15:25–38PubMedGoogle Scholar
  3. 3.
    Rosadas C, Cabral-Castro MJ, Vicente AC et al (2013) Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells. J Virol Methods 193:536–541CrossRefPubMedGoogle Scholar
  4. 4.
    Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 50–30 exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88: 7276–7280CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994CrossRefPubMedGoogle Scholar
  6. 6.
    Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154CrossRefPubMedGoogle Scholar
  7. 7.
    Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582CrossRefPubMedGoogle Scholar
  8. 8.
    Qiagen (2010) Critical factors for successful real-time PCR. Accessed 9 Nov 2013
  9. 9.
    Yu Y, Lee C, Kim J et al (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670–679CrossRefPubMedGoogle Scholar
  10. 10.
    Raymaekers M, Smets R, Maes B et al (2009) Checklist for optimization and validation of real-time PCR assays. J Clin Lab Anal 23:145–151CrossRefPubMedGoogle Scholar
  11. 11.
    Lim J, Shin SG, Lee S et al (2011) Design and use of group-specific primers and probes for real-time quantitative PCR. Front Environ Sci Eng 5:28–39CrossRefGoogle Scholar
  12. 12.
    Chuang LY, Cheng YH, Yang CH (2013) Specific primer design for the polymerase chain reaction. Biotechnol Lett 35:1541–1549CrossRefPubMedGoogle Scholar
  13. 13.
    Hanna SE, Connor CJ, Wang HH (2005) Real-time polymerase chain reaction for the food microbiologist: technologies, applications, and limitations. J Food Sci 70:49–53CrossRefGoogle Scholar
  14. 14.
    Toouli CD, Turner DR, Grist SA et al (2000) The effect of cycle number and target size on polymerase chain reaction amplification of polymorphic repetitive sequences. Anal Biochem 280:324–326CrossRefPubMedGoogle Scholar
  15. 15.
    McConlogue L, Brow MA, Innis MA (1988) Structure-independent DNA amplification by PCR using 7-deaza-20-deoxyguanosine. Nucleic Acids Res 16:9869CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Mitsuhashi M (1996) Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences. J Clin Lab Anal 10:277–284CrossRefPubMedGoogle Scholar
  17. 17.
    Wittwer CT, Herrmann MG, Moss AA et al (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–131PubMedGoogle Scholar
  18. 18.
    Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160CrossRefPubMedGoogle Scholar
  19. 19.
    Wu JS, Lee C, Wu CC et al (2004) Primer design using genetic algorithm. Bioinformatics 20:1710–1717CrossRefPubMedGoogle Scholar
  20. 20.
    Marchesi JR (2001) Primer design for PCR amplification of environmental DNA targets. In: Rochelle PA (ed) Environmental molecular microbiology: protocols and applications. Horizon Scientific Press, Wymondham, pp 43–54Google Scholar
  21. 21.
    Simonsson T, Pecinka P, Kubista M (1998) DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res 26:1167–1172CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Giulietti A, Overbergh L, Valckx D et al (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386–401CrossRefPubMedGoogle Scholar
  23. 23.
    Gunson RN, Collins TC, Carman WF (2006) Practical experience of high throughput real time PCR in the routine diagnostic virology setting. J Clin Virol 35:355–367CrossRefPubMedGoogle Scholar
  24. 24.
    Saiki RK (1989) The design and optimization of the PCR. In: Erlich HA (ed) PCR technology: principles and applications for DNA amplification. McMillan Publishers (Stockton Press), New York, NY, pp 7–22Google Scholar
  25. 25.
    Kubista M, Andrade JM, Bengtsson M et al (2006) The real-time polymerase chain reaction. Mol Asp Med 27:95–125CrossRefGoogle Scholar
  26. 26.
    Polz MF, Cavanaugh CM (1998) Bias in template-to-product rations in multitemplate PCR. Appl Environ Microbiol 64:3724–3730PubMedCentralPubMedGoogle Scholar
  27. 27.
    Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456CrossRefPubMedGoogle Scholar
  28. 28.
    Biorad (2013) qPCR assay design and optimization. Accessed 24 Oct 2013
  29. 29.
    Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144CrossRefPubMedGoogle Scholar
  30. 30.
    Abd-Elsalam KA (2003) Bioinformatic tools and guideline for PCR primer design. Afr J Biotechnol 2:91–95CrossRefGoogle Scholar
  31. 31.
    Fredman D, Jobs M, Strömqvist L et al (2004) DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications. Hum Mutat 24:1–8CrossRefPubMedGoogle Scholar
  32. 32.
    Nonis A, Scortegagna M, Nonis A et al (2011) PRaTo: a web-tool to select optimal primer pairs for qPCR. Biochem Biophys Res Commun 415:707–708CrossRefPubMedGoogle Scholar
  33. 33.
    Gubelmann C, Gattiker A, Massouras A et al (2011) GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR. Database 2011:bar040. doi: 10.1093/database/bar040 CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Rychlik W (2007) OLIGO 7 primer analysis software. In: Yuryev A (ed) Methods in molecular biology, vol 402, PCR primer design. Humana, Totowa, NJ, pp 35–59Google Scholar
  35. 35.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar
  36. 36.
    Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3: new capabilities and interfaces. Nucleic Acids Res 40:e115CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Untergasser A, Nijveen H, Rao X et al (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472CrossRefPubMedGoogle Scholar
  39. 39.
    Marshall OJ (2007) Graphical design of primers with PerlPrimer. In: Yuryev A (ed) Methods in molecular biology, vol 402, PCR primer design. Humana, Totowa, NJ, pp 403–414Google Scholar
  40. 40.
    Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20:2399–2400CrossRefPubMedGoogle Scholar
  41. 41.
    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  42. 42.
    Arvidsson S, Kwasniewski M, Riaño-Pachón DM et al (2008) QuantPrime: a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinformatics 9:465CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Ziesel AC, Chrenek MA, Wong PW (2008) MultiPriDe: automated batch development of quantitative real-time PCR primers. Nucleic Acids Res 36:3095–3100CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Vijaya SR, Kumar K, Zavaljevski N et al (2010) A high-throughput pipeline for the design of real-time PCR signatures. BMC Bioinformatics 11:340CrossRefGoogle Scholar
  45. 45.
    Brosseau JP, Lucier JF, Lapointe E et al (2010) High-throughput quantification of splicing isoforms. RNA 16:442–449CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Sobhy H, Colson P (2012) Gemi: PCR primers prediction from multiple alignments. Comp Funct Genomics 2012:783138. doi: 10.1155/2012/783138 CrossRefPubMedCentralPubMedGoogle Scholar
  47. 47.
    Brodin J, Krishnamoorthy M, Athreya G et al (2013) A multiple-alignment based primer design algorithm for genetically highly variable DNA targets. BMC Bioinformatics 14:255CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    Applied Biosystems (2004) Primer Express software version 3.0. getting started guide. Accessed 10 Jan 2005
  49. 49.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedCentralPubMedGoogle Scholar
  50. 50.
    You FM, Huo N, Gu YQ et al (2009) ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery. BMC Bioinformatics 10:331CrossRefPubMedCentralPubMedGoogle Scholar
  51. 51.
    You FM, Huo N, Gu YQ et al (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253CrossRefPubMedCentralPubMedGoogle Scholar
  52. 52.
    Riaz T, Shehzad W, Viari A et al (2011) ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis. Nucleic Acids Res 39:e145CrossRefPubMedCentralPubMedGoogle Scholar
  53. 53.
    Wu X, Munroe DJ (2006) EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 5:119–120CrossRefPubMedGoogle Scholar
  54. 54.
    Cao Y, Sun J, Zhu J et al (2010) PrimerCE: designing primers for cloning and gene expression. Mol Biotechnol 46:113–117CrossRefPubMedGoogle Scholar
  55. 55.
    Lefever S, Vandesompele J, Speleman F et al (2009) RTPrimerDB: the portal for real-time PCR primers and probes. Nucleic Acids Res 37:D942–D945CrossRefPubMedCentralPubMedGoogle Scholar
  56. 56.
    Fredslund J (2008) DATFAP: a database of primers and homology alignments for transcription factors from 13 plant species. BMC Genomics 9:140CrossRefPubMedCentralPubMedGoogle Scholar
  57. 57.
    Wang X, Spandidos A, Wang H et al (2012) PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res 40:D1144–D1149CrossRefPubMedCentralPubMedGoogle Scholar
  58. 58.
    Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics 3:1–14Google Scholar
  59. 59.
    Guerrero D, Bautista R, Villalobos DP et al (2010) AlignMiner: a web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences. Algorithms Mol Biol 5:24CrossRefPubMedCentralPubMedGoogle Scholar
  60. 60.
    Taylor S, Wkem M, Dijkman G et al (2010) A practical approach to RT-qPCR: publishing data that conform to the MIQE guidelines. Methods 50:S1–S5CrossRefPubMedGoogle Scholar
  61. 61.
    Lam CW, Mak CM (2013) Allele dropout caused by a non-primer-site SNV affecting PCR amplification: a call for next-generation primer design algorithm. Clin Chim Acta 421:208–212CrossRefPubMedGoogle Scholar
  62. 62.
    Karlin S, Altschul SF (1990) Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci U S A 87:2264–2268CrossRefPubMedCentralPubMedGoogle Scholar
  63. 63.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  64. 64.
    Mallona I, Weiss J, Egea-Cortines M (2011) pcrEfficiency: a web tool for PCR amplification efficiency prediction. BMC Bioinformatics 12:404CrossRefPubMedCentralPubMedGoogle Scholar
  65. 65.
    Edwards KJ (2004) Performing real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 71–83Google Scholar
  66. 66.
    Applied Biosystems (2010) Real-time PCR systems. Reagent guide. Accessed 7 Jul 2010
  67. 67.
    Promega Corporation (2009) Protocols & applications guide. Accessed 21 Oct 2013
  68. 68.
    Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) A-Z of Quantitative PCR (IUL Biotechnology, No. 5). International University Line (IUL), San Diego, CA, pp 87–112Google Scholar
  69. 69.
    Lee MA, Squirell DJ, Leslie DL et al (2004) Homogeneous fluorescent chemistries for real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 31–70Google Scholar
  70. 70.
    Life Technologies Corporation (2012) Real-time PCR handbook. Accessed 6 Nov 2013
  71. 71.
    Rajeevan MS, Ranamukhaarachchi DG, Vernon SD et al (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25:443–451CrossRefPubMedGoogle Scholar
  72. 72.
    Kavanagh I, Jones G, Nayab SN (2011) Significance of controls and standard curves in PCR. In: Kennedy S, Oswald N (eds) PCR troubleshooting and optimization: the essential guide. Caister Academic Press, Norfolk, pp 67–78Google Scholar
  73. 73.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 1:29–45CrossRefGoogle Scholar
  74. 74.
    Gadkar VY, Filion M (2013) New developments in quantitative real-time polymerase chain reaction technology. Curr Issues Mol Biol 8:1–6Google Scholar
  75. 75.
    Ishii T, Sootome H, Shan L et al (2007) Validation of universal conditions for duplex quantitative reverse transcription polymerase chain reaction assays. Anal Biochem 362:201–212CrossRefPubMedGoogle Scholar
  76. 76.
    Quellhorst, G., Rulli, S. (2008) A systematic guideline for developing the best real-time PCR primers. SABiosci. Accessed 26 Aug 2013
  77. 77.
    Bustin SA, Nolan T (2004) Analysis of mRNA expression by real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 125–184Google Scholar
  78. 78.
    Zhang J, Byrne CD (1999) Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. Biochem J 337:231–241CrossRefPubMedCentralPubMedGoogle Scholar
  79. 79.
    Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM et al (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307:63–69CrossRefPubMedGoogle Scholar
  80. 80.
    VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626CrossRefPubMedGoogle Scholar
  81. 81.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  82. 82.
    Wang X, Seed B (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res 31:e154CrossRefPubMedCentralPubMedGoogle Scholar
  83. 83.
    Applied Biosystems (2008) Guide to performing relative quantitation of gene expression using real-time quantitative PCR. Accessed 2 Jun 2008
  84. 84.
    Bauer P, Rolfs A, Regitz-Zagrosek V et al (1997) Use of manganese in RT-PCR eliminates PCR artefacts resulting from DNase I digestion. Biotechniques 22:1128–1132PubMedGoogle Scholar
  85. 85.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193CrossRefPubMedGoogle Scholar
  86. 86.
    Rodríguez A (2012) Desarrollo de métodos de PCR en tiempo real para la detección y cuantificación de mohos productores de micotoxinas en alimentos. Doctoral Thesis. University of Extremadura, SpainGoogle Scholar
  87. 87.
    Sayers EW, Barrett T, Benson DA et al (2012) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 40:D13–D25CrossRefPubMedCentralPubMedGoogle Scholar
  88. 88.
    Cui W, Taub DD, Gardner K (2007) qPrimerDepot: a primer database for quantitative real time PCR. Nucleic Acids Res 35:D805–D809CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Alicia Rodríguez
    • 1
  • Mar Rodríguez
    • 1
  • Juan J. Córdoba
    • 1
  • María J. Andrade
    • 1
    Email author
  1. 1.Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary ScienceUniversity of ExtremaduraCáceresSpain

Personalised recommendations