title: The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.
abstract: BACKGROUND
  The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid.
  
  
  RESULTS
  The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes.
  
  
  CONCLUSIONS
  Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors.forename: Meesbah
authors.initials: M
authors.lastname: Jiwaji
authors.forename: Rónán
authors.initials: R
authors.lastname: Daly
authors.forename: Kshama
authors.initials: K
authors.lastname: Pansare
authors.forename: Pauline
authors.initials: P
authors.lastname: McLean
authors.forename: Jingli
authors.initials: J
authors.lastname: Yang
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Pitt
mesh_terms: D000818:Animals; D002875:Chromosomes / Q000235:genetics; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics* / Q000378:metabolism / Q000592:standards; D016133:Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D012984:Software; D014162:Transfection
chemical_list: D049410:Luciferases, Renilla