背景 视网膜新生血管常伴随微小RNA(miRNA)表达水平的变化.实时定量PCR是miRNA表达谱分析的常用方法,但内参基因在不同实验条件下可能呈现出差异性表达,如果以表达水平有显著波动的管家基因作为内参基因,可能会影响目的基因表达的结果评估,确定稳定表达的内参基因是进行后续研究的前提. 目的 比较氧诱导视网膜病变(OIR)模型小鼠眼球组织中常用的miRNA内参基因及其他几种内参基因表达水平的差异及稳定性,筛选在OIR模型和正常发育条件下均稳定表达的最佳miRNA内参基因.方法 应用随机数字表法抽取正常清洁级C57BL/6J P7、P12、P17、P37不同日龄小鼠及成年鼠(8周龄)各10只,小鼠在相应时间点颈椎脱臼法处死,收集眼球组织,应用实时定量PCR技术检测用于评估核小分子RNAU6(RNU6)、5S核糖体RNA(5s rRNA)、核仁小分子RNA U68(U68)、核仁小分子RNA U70(U70)、核仁小分子RNA U49A(U49A)5种内参基因在正常发育的不同日龄(P7、P12、P17、P37及成年)小鼠中表达的动态变化;另选取P7幼鼠40只,采用随机数字表法随机分为OIR组和正常对照组,每组20只.正常对照组小鼠在正常氧环境下饲养,OIR组小鼠在体积分数(75±2)％的高氧环境中饲养10d.分别选取P17 OIR组和正常对照组小鼠各5只,进行球后静脉荧光素视网膜血管造影,并制备视网膜铺片,另各取5只小鼠处死后制备视网膜切片行组织病理学检查,以验证是否造模成功.然后应用实时定量PCR技术检测上述5种内参基因在正常对照组和OIR组小鼠视网膜组织中的表达差异;应用GeNorm程序对内参基因表达的稳定性进行分析. 结果 在不同日龄正常小鼠视网膜中,内参基因U68的表达水平变化最大,其次为U49A、U70、RNU6、5s rRNA;内参基因U68、U49A、U70、RNU6在不同日龄小鼠视网膜中的相对表达量的总体差异均有统计学意义(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),而5s rRNA在不同日龄小鼠视网膜中的相对表达量差异无统计学意义(F=2.099,P=0.107).P17 OIR组小鼠视网膜铺片显示,视网膜血管迂曲,可见视网膜无灌注区,苏木精-伊红染色可见突破视网膜内界膜的新生血管芽,而正常对照组小鼠视网膜均正常.在P17 OIR组小鼠内参基因U70表达水平变化最大,其次为U49A、U68、5s rRNA;U70、U49A、U68、5s rRNA在OIR组和正常对照组小鼠视网膜中表达量的差异均有统计学意义(t=5.174,P=0.000;t=3.376,P=0.012;t=4.802,P=0.000;t=2.856,P=0.029),RNU6的相对表达量在2个组间的差异无统计学意义(t=2.104,P=0.065).GeNorm程序分析结果显示,不同日龄的正常小鼠眼球中所选内参基因表达稳定度为5s rRNA/RNU6＞ U70＞ U49A＞ U68,最佳内参基因组合为5s rRNA和RNU6;在P17OIR组小鼠眼球组织中所选内参基因的表达稳定度为5s rRNA/RNU6＞ U68＞ U49A＞ U70,最佳内参基因组合亦为5s rRNA和RNU6.结论 应用实时定量PCR技术研究miRNA基因表达时,应根据不同的研究对象和处理条件选择合适的内参基因.在OIR模型中,综合考虑发育、缺氧等因素的影响,在条件允许的情况下可优先选择5s rRNA和RNU6组合作为小鼠眼球组织miRNA表达分析的内参基因.%Background 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.Objective 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.Methods 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.Results 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.Conclusions 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.
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