Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells

Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms.We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.Gene expression analysis is important in the identification of new biological and disease mechanisms. Real-time reverse transcription quantitative polymerase chain reaction (qRT-PCR) is one of the most widely applied methods to measure transcript abundance. Whether the results are truly reflective of biological processes is dependent on normalisation of data against stable endogenous controls, often referred to as housekeeping genes. A housekeeping gene is a constitutively expressed gene that is expressed in all cells of an organism [1]. An ideal housekeeping gene should be expressed at the same level in different types of cells [2]. However, whilst some of the reference genes are expressed at relatively constant levels, others may vary depending on the experimental conditions, [3] and sample type and quality [4]. A number of reports indicate that no housekeeping gene can be considered to be suitable for all condit