Conclusion

qRT-PCR has revolutionized RNA expression measurement that led to an explosion of publications over the last 8 years. Ironically, acceptable normalization methods, particularly with reference genes, have remained a contentious issue and it has only been over the last 4 years that this has been addressed. Despite this, even today RNA expression data are published with no mention of whether the methods used for normalization have been validated. However, without this validation the assay may not have the resolution to detect small changes or worse produce artefactual results. Worryingly, papers are still being published even though the authors have not demonstrated any validation of their normalization strategies.

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