Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Diabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.