Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references

Abstract

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

Keywords

Gene expression; normalization; quantitative RT-PCR; reference gene; transcript quantification

Published in

Journal of Experimental Botany
2009, volume: 60, number: 2, pages: 487-493

SLU Authors

• Mauriat, Melanie

  • Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences
    

• Bellini, Catherine

  • Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences
    
  • National Institute of Agricultural Research (INRA)

UKÄ Subject classification

Forest Science

Publication identifier

• DOI: https://doi.org/10.1093/jxb/ern305

Permanent link to this page (URI)

https://res.slu.se/id/publ/28559