The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells

Hdl Handle:
http://hdl.handle.net/10147/315466
Title:
The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells
Authors:
Jiwaji, Meesbah; Daly, Rónán; Pansare, Kshama; McLean, Pauline; Yang, Jingli; Kolch, Walter; Pitt, Andrew R
Citation:
BMC Molecular Biology. 2010 Dec 31;11(1):103
Issue Date:
31-Dec-2010
URI:
http://dx.doi.org/10.1186/1471-2199-11-103; http://hdl.handle.net/10147/315466
Abstract:
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.
Item Type:
Article
Language:
en
Keywords:
GENETICS
Local subject classification:
CELL BIOLOGY

Full metadata record

DC FieldValue Language
dc.contributor.authorJiwaji, Meesbahen_GB
dc.contributor.authorDaly, Rónánen_GB
dc.contributor.authorPansare, Kshamaen_GB
dc.contributor.authorMcLean, Paulineen_GB
dc.contributor.authorYang, Jinglien_GB
dc.contributor.authorKolch, Walteren_GB
dc.contributor.authorPitt, Andrew Ren_GB
dc.date.accessioned2014-04-07T10:03:26Z-
dc.date.available2014-04-07T10:03:26Z-
dc.date.issued2010-12-31-
dc.identifier.citationBMC Molecular Biology. 2010 Dec 31;11(1):103en_GB
dc.identifier.urihttp://dx.doi.org/10.1186/1471-2199-11-103-
dc.identifier.urihttp://hdl.handle.net/10147/315466-
dc.description.abstractAbstract 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.-
dc.language.isoenen
dc.subjectGENETICSen_GB
dc.subject.otherCELL BIOLOGYen_GB
dc.titleThe Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cellsen_GB
dc.typeArticleen
dc.language.rfc3066en-
dc.rights.holderMeesbah Jiwaji et al.; licensee BioMed Central Ltd.-
dc.description.statusPeer Reviewed-
dc.date.updated2014-04-03T04:26:46Z-
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