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

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. © 2010 Jiwaji et al; licensee BioMed Central Ltd.

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest. © 2011 Elsevier Inc.

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type I diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. in contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright (c) 2009 John Wiley & Sons, Ltd.

RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript. © 2003 Elsevier Ltd. All rights reserved.