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.
揹景 視網膜新生血管常伴隨微小RNA(miRNA)錶達水平的變化.實時定量PCR是miRNA錶達譜分析的常用方法,但內參基因在不同實驗條件下可能呈現齣差異性錶達,如果以錶達水平有顯著波動的管傢基因作為內參基因,可能會影響目的基因錶達的結果評估,確定穩定錶達的內參基因是進行後續研究的前提. 目的 比較氧誘導視網膜病變(OIR)模型小鼠眼毬組織中常用的miRNA內參基因及其他幾種內參基因錶達水平的差異及穩定性,篩選在OIR模型和正常髮育條件下均穩定錶達的最佳miRNA內參基因.方法 應用隨機數字錶法抽取正常清潔級C57BL/6J P7、P12、P17、P37不同日齡小鼠及成年鼠(8週齡)各10隻,小鼠在相應時間點頸椎脫臼法處死,收集眼毬組織,應用實時定量PCR技術檢測用于評估覈小分子RNAU6(RNU6)、5S覈糖體RNA(5s rRNA)、覈仁小分子RNA U68(U68)、覈仁小分子RNA U70(U70)、覈仁小分子RNA U49A(U49A)5種內參基因在正常髮育的不同日齡(P7、P12、P17、P37及成年)小鼠中錶達的動態變化;另選取P7幼鼠40隻,採用隨機數字錶法隨機分為OIR組和正常對照組,每組20隻.正常對照組小鼠在正常氧環境下飼養,OIR組小鼠在體積分數(75±2)％的高氧環境中飼養10d.分彆選取P17 OIR組和正常對照組小鼠各5隻,進行毬後靜脈熒光素視網膜血管造影,併製備視網膜鋪片,另各取5隻小鼠處死後製備視網膜切片行組織病理學檢查,以驗證是否造模成功.然後應用實時定量PCR技術檢測上述5種內參基因在正常對照組和OIR組小鼠視網膜組織中的錶達差異;應用GeNorm程序對內參基因錶達的穩定性進行分析. 結果 在不同日齡正常小鼠視網膜中,內參基因U68的錶達水平變化最大,其次為U49A、U70、RNU6、5s rRNA;內參基因U68、U49A、U70、RNU6在不同日齡小鼠視網膜中的相對錶達量的總體差異均有統計學意義(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在不同日齡小鼠視網膜中的相對錶達量差異無統計學意義(F=2.099,P=0.107).P17 OIR組小鼠視網膜鋪片顯示,視網膜血管迂麯,可見視網膜無灌註區,囌木精-伊紅染色可見突破視網膜內界膜的新生血管芽,而正常對照組小鼠視網膜均正常.在P17 OIR組小鼠內參基因U70錶達水平變化最大,其次為U49A、U68、5s rRNA;U70、U49A、U68、5s rRNA在OIR組和正常對照組小鼠視網膜中錶達量的差異均有統計學意義(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的相對錶達量在2箇組間的差異無統計學意義(t=2.104,P=0.065).GeNorm程序分析結果顯示,不同日齡的正常小鼠眼毬中所選內參基因錶達穩定度為5s rRNA/RNU6＞ U70＞ U49A＞ U68,最佳內參基因組閤為5s rRNA和RNU6;在P17OIR組小鼠眼毬組織中所選內參基因的錶達穩定度為5s rRNA/RNU6＞ U68＞ U49A＞ U70,最佳內參基因組閤亦為5s rRNA和RNU6.結論 應用實時定量PCR技術研究miRNA基因錶達時,應根據不同的研究對象和處理條件選擇閤適的內參基因.在OIR模型中,綜閤攷慮髮育、缺氧等因素的影響,在條件允許的情況下可優先選擇5s rRNA和RNU6組閤作為小鼠眼毬組織miRNA錶達分析的內參基因.
배경 시망막신생혈관상반수미소RNA(miRNA)표체수평적변화.실시정량PCR시miRNA표체보분석적상용방법,단내삼기인재불동실험조건하가능정현출차이성표체,여과이표체수평유현저파동적관가기인작위내삼기인,가능회영향목적기인표체적결과평고,학정은정표체적내삼기인시진행후속연구적전제. 목적 비교양유도시망막병변(OIR)모형소서안구조직중상용적miRNA내삼기인급기타궤충내삼기인표체수평적차이급은정성,사선재OIR모형화정상발육조건하균은정표체적최가miRNA내삼기인.방법 응용수궤수자표법추취정상청길급C57BL/6J P7、P12、P17、P37불동일령소서급성년서(8주령)각10지,소서재상응시간점경추탈구법처사,수집안구조직,응용실시정량PCR기술검측용우평고핵소분자RNAU6(RNU6)、5S핵당체RNA(5s rRNA)、핵인소분자RNA U68(U68)、핵인소분자RNA U70(U70)、핵인소분자RNA U49A(U49A)5충내삼기인재정상발육적불동일령(P7、P12、P17、P37급성년)소서중표체적동태변화;령선취P7유서40지,채용수궤수자표법수궤분위OIR조화정상대조조,매조20지.정상대조조소서재정상양배경하사양,OIR조소서재체적분수(75±2)％적고양배경중사양10d.분별선취P17 OIR조화정상대조조소서각5지,진행구후정맥형광소시망막혈관조영,병제비시망막포편,령각취5지소서처사후제비시망막절편행조직병이학검사,이험증시부조모성공.연후응용실시정량PCR기술검측상술5충내삼기인재정상대조조화OIR조소서시망막조직중적표체차이;응용GeNorm정서대내삼기인표체적은정성진행분석. 결과 재불동일령정상소서시망막중,내삼기인U68적표체수평변화최대,기차위U49A、U70、RNU6、5s rRNA;내삼기인U68、U49A、U70、RNU6재불동일령소서시망막중적상대표체량적총체차이균유통계학의의(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),이5s rRNA재불동일령소서시망막중적상대표체량차이무통계학의의(F=2.099,P=0.107).P17 OIR조소서시망막포편현시,시망막혈관우곡,가견시망막무관주구,소목정-이홍염색가견돌파시망막내계막적신생혈관아,이정상대조조소서시망막균정상.재P17 OIR조소서내삼기인U70표체수평변화최대,기차위U49A、U68、5s rRNA;U70、U49A、U68、5s rRNA재OIR조화정상대조조소서시망막중표체량적차이균유통계학의의(t=5.174,P=0.000;t=3.376,P=0.012;t=4.802,P=0.000;t=2.856,P=0.029),RNU6적상대표체량재2개조간적차이무통계학의의(t=2.104,P=0.065).GeNorm정서분석결과현시,불동일령적정상소서안구중소선내삼기인표체은정도위5s rRNA/RNU6＞ U70＞ U49A＞ U68,최가내삼기인조합위5s rRNA화RNU6;재P17OIR조소서안구조직중소선내삼기인적표체은정도위5s rRNA/RNU6＞ U68＞ U49A＞ U70,최가내삼기인조합역위5s rRNA화RNU6.결론 응용실시정량PCR기술연구miRNA기인표체시,응근거불동적연구대상화처리조건선택합괄적내삼기인.재OIR모형중,종합고필발육、결양등인소적영향,재조건윤허적정황하가우선선택5s rRNA화RNU6조합작위소서안구조직miRNA표체분석적내삼기인.
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.
