Validation of RT-qPCR reference genes and determination of Robo4 expression levels in human retinal endothelial cells under hypoxia and/or hyperglycemia.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the most common technique to investigate mRNA expression levels of target genes. In order to obtain accurate results, stable reference genes need to be selected for normalization in an experimental study. Human retinal endothelial cells (HREC) cultured in a hypoxic and hyperglycemic environment is a potential cell model to study diabetic retinopathy (DR), but the proper reference genes for RNA analysis have not yet been determined. In the present study, we evaluated the expression levels of 14 candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR for HREC under hypoxic and/or hyperglycemic conditions. The results of the analyses using GeNorm, NormFinder, and BestKeeper software showed that a combination of TBP, PUM1, and ALAS1 was most suitable for this research. Based on these results, mRNA expression levels of Roundabout4 (Robo4) in HREC were determined. The RT-qPCR analysis showed that there was a significant increase in Robo4 expression under hyperglycemic conditions, while there was a decrease in expression under hypoxic and combined hypoxic and hyperglycemic conditions, suggesting that Robo4 might play different roles in various stages of DR.