REVIEW-ARTICLE |
Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references
1Molecular Biology Centre, Centre de Ressources Régionales en Biologie Moléculaire, Université de Picardie Jules Verne, Faculté des Sciences, 33 Rue St Leu, F-80039 Amiens, France
2Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden
3EA3900 Biologie des plantes et contrôle des insectes ravageurs, Université de Picardie Jules Verne, Faculté des Sciences, 33 Rue St Leu, F-80039 Amiens, France
4Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, F-78026 Versailles Cedex, France
* To whom correspondence should be addressed: E-mail: laurent.gutierrez{at}u-picardie.fr
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.
Key words: Gene expression, normalization, quantitative RT-PCR, reference gene, transcript quantification
Received 8 July 2008; Revised 31 October 2008 Accepted 10 November 2008