Optimization of reference genes for microRNA expression analysis in a mouse model of oxygen-induced retinopathy

Retinal neovascularization varies with the change of microRNA (miRNA) expression level.Quantitative real-time PCR (qRT-PCR) is a common method for the analysis of miRNA expression profiling.However,the housekeeping genes selected as references may exhibit differential expression levels under the distinctive experimental conditions.The accuracy of the levels of target gene expression often is affected if the selected housekeeping genes with significantly fluctuating expression as references.Determining a reference gene with stable expression level is the premise to consecutive studies.

This study was to compare the expression levels and stability of the frequently used reference genes for miRNA expression analysis in normally developing eyes and in eyes of a mouse model of oxygen-induced retinopathy (OIR),and select the optimal reference gene(s) exhibiting stable ocular expression under both hypoxia and normal development conditions.

P7,P12,P17,P37 and 8-week-old clean C57BL/6J mice were selected randomly.q-PCR was used to detect the dynamic changes of relative expressions of 5 kinds of reference genes in different ages of mice,including snRNAU6 (RNU6),5S ribosomal RNA (5s rRNA),snoRNA U68 (U68),snoRNA U70 (U70),snoRNA U49A (U49A).Other 40 P7 mice were randomized into the normal control group and the OIR group.The mice of the normal control group were fed in the normal oxygen environment,and those in the OIR group were raised in the (75±2)% oxygen environment for 10 days.Fluorescine was injected via ritrobulbar vein for retinal stretched preparation,and the histopathological examination of mouse retinas was performed to identified the models.The expressing difference of 5 kinds of reference genes in the retinas were detected to compare the differences of 5 kinds of genes expression between the two groups.GeNorm program was employed to compare the stability of the expressing genes.This study were approved and granted by Ethical Committee of Tianjin Medical University and met the requirements stipulated in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Across the developmental ages,the expression levels of U68 in the eye exhibited the greatest variability,and then coming with U49A,U70,RNU6 and 5s rRNA.Significant differences were seen across the developmental ages for the expression levels of U68,U49A,U70,and RNU6 (U70:F=7.005,P=0.000;U68:F=10.189,P=0.000;U49A:F=21.134,P=0.000;RNU6:F=4.968,P=0.004).Expression of 5s rRNA showed the least variability across the developmental ages (F=2.099,P=0.107).In P17 mice of the OIR group,tortuous access vessels and non-perfusion area were found in the retinal section.Hematoxylin-eosin stain showed the neovascular sprout through across the inner limiting membrane.The relative expression of U70 exhibited the greatest variability and then were U49A,U68 and 5s rRNA in turn in P17 OIR mice,and significantly elevated expressions were found in U70,U49A,U68 and 5s rRNA genes between the OIR group and the normal control group (t=5.174,P=0.000;t=3.376,P=0.012;t=4.802,P=0.000;t=2.856, P=0.029).Expression of RNU6 showed the least variability between the two groups (t=2.104,P=0.065).As analyzed by GeNorm program,the stability for the five reference genes across developmental ages was 5s rRNA/RNU6>U70>U49A>U68,and the optimal reference combination was 5s rRNA and RNU6.Whereas the stability for the OIR model was 5s rRNA/RNU6>U68>U49A>U70,and the optimal reference combination was 5s rRNA and RNU6.

Reference genes should be selected based on specific subjects and experimental conditions when qRT-PCR is used to analyze miRNA expression levels. In the OIR model, both developmental and hypoxic factors need to be considered. 5s rRNA and RNU6 reference combination is the preferred option for the qRT-PCR analysis of ocular miRNA expression if available.