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

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

Abstract

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 

Notes

Acknowledgments

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.

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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

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