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

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionsOur 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.

Unique Reporter-Based Sensor Platforms to Monitor Signalling in Cells

“…[20]. HEK293 cells (ATCC Number CRL-1573) were maintained in DMEM supplemented with 4 mM L-glutamine and 10% FBS at 37°C in an atmosphere that contained 5% CO 2 .…”

“…Primer sequences are shown in Tables S1 and S2. A 10 fold dilution series of UR or Rluc linear dsDNA was created and a standard curve generated as described before [20]–[21]. Efficiency of the qPCR reaction (E) was calculated and primer pairs with E = 1.6–2.4 were typically used [22].…”

“…Unknown samples were compared to the standard curve and the copy number calculated. Transfection efficiency was accounted for by normalizing the UR copy numbers to that of Rluc in each sample [20]. Changes in gene expression were quantified by comparing the log2 ratio for treated cells to untreated cells.…”

In vivo Bioluminescence Imaging Monitoring of Stem Cells' Participation in Choroidal Neovascularization

“…In future research, mechanisms of stem cell participation in CNV should be a research priority. Finally, we used Fluc in the present study and more types of luciferase could be used to observe cell behavior in the future [29,30]. In addition, the CNV model has been used in most studies on CNV, and the mechanism of the CNV model and which patients are similar [15,16], but we have to admit that there are some defects in the CNV model, because the animal model is an acute one, whereas the patients are chronic cases, and the study which optimizes the CNV model will be our major work in the future.…”

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

“…Data analysis was performed using 2 -ΔΔCt method (18). The results were normalized against β-actin expression level as a reference gene (19). All the reactions were performed in triplicate.…”