title: NORMA-Gene: a simple and robust method for qPCR normalization based on target gene data.
abstract: BACKGROUND
  Normalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and to validate.
  
  
  RESULTS
  We developed a data driven normalization algorithm (NORMA-Gene). An analysis of the performance of NORMA-Gene compared to reference gene normalization on artificially generated data-sets showed that the NORMA-Gene normalization yielded more precise results under a large range of parameters tested. Furthermore, when tested on three very different real qPCR data-sets NORMA-Gene was shown to be best at reducing variance due to experimental bias in all three data-sets compared to normalization based on the use of reference gene(s).
  
  
  CONCLUSIONS
  Here we present the NORMA-Gene algorithm that is applicable to all biological and biomedical qPCR studies, especially those that are based on a limited number of assayed genes. The method is based on a data-driven normalization and is useful for as little as five target genes comprising the data-set. NORMA-Gene does not require the identification and validation of reference genes allowing researchers to focus their efforts on studying target genes of biological relevance.
authors.affiliation: National Environmental Research Institute, Aarhus University, Department of Terrestrial Ecology, Vejlsøvej 25, DK-8600 Silkeborg, Denmark.
authors.forename: Lars-Henrik
authors.initials: LH
authors.lastname: Heckmann
authors.forename: Peter B
authors.initials: PB
authors.lastname: Sørensen
authors.forename: Paul Henning
authors.initials: PH
authors.lastname: Krogh
authors.forename: Jesper G
authors.initials: JG
authors.lastname: Sørensen
mesh_terms: D000465:Algorithms; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*