Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references
Open Access
- 6 January 2009
- journal article
- review article
- Published by Oxford University Press (OUP) in Journal of Experimental Botany
- Vol. 60 (2), 487-493
- https://doi.org/10.1093/jxb/ern305
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
This publication has 27 references indexed in Scilit:
- Towards a Systematic Validation of References in Real-Time RT-PCRPlant Cell, 2008
- The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plantsPlant Biotechnology Journal, 2008
- A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factorsPlant Methods, 2007
- Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in ArabidopsisPlant Physiology, 2005
- The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalizationAnalytical Biochemistry, 2005
- Quantitative real-time RT-PCR – a perspectiveJournal of Molecular Endocrinology, 2005
- Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR – how well do they correlate?BMC Genomics, 2005
- Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data SetsCancer Research, 2004
- Real‐time RT‐PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root‐ and shoot‐specific genesThe Plant Journal, 2004
- Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA LevelsJournal of Bacteriology, 2001