Research article - Peer-reviewed, 2009
Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references
Guenin, Stephanie; Mauriat, Melanie; Pelloux, Jerome; Van Wuytswinkel, Olivier; Bellini, Catherine; Gutierrez, LaurentAbstract
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 quantificationPublished in
Journal of Experimental Botany2009, volume: 60, number: 2, pages: 487-493
Authors' information
Guenin, Stephanie
Mauriat, Melanie
Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology
Pelloux, Jerome
van Wuytswinkel, Olivier
Bellini, Catherine
Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology
Bellini, Catherine
National Institute of Agricultural Research (INRA)
UKÄ Subject classification
Forest Science
Publication Identifiers
DOI: https://doi.org/10.1093/jxb/ern305
URI (permanent link to this page)
https://res.slu.se/id/publ/28559