脑脊液miRNA定量检测中内参基因的选择评价

The optimal reference genes for miRNA quantitative analysis in cerebrospinal fluid of patients with tuberculous meningitis

目的:探讨常规内参基因U6和Cel-miR-39用于结核性脑膜炎患者脑脊液微小核糖核酸(miRNA)定量检测的可行性,并初步用于脑脊液miRNA定量检测分析。方法:本研究选取首都医科大学附属北京胸科医院常规脑脊液检测后保存的标本,根据最终诊断依据纳入结核性脑膜炎患者36例、病毒性脑膜炎患者34例。提取其脑脊液标本miRNA,采用TaqMan探针法检测miRNA的表达水平。采用GeNorm、NormFinder和Bestkeeper软件分析内参基因miRNA的稳定性,采用2-ΔCt方法计算靶标miRNA的相对表达量。标本重复检测的一致性分析采用Pearson检验。靶标基因在两组间的表达差异比较采用t检验。结果:70份脑脊液标本中,U6检测的循环阈值(Ct)为(30.40±3.30)个循环,表达丰度较低,Cel-miR-39检测的C t值为(21.49±0.70)个循环,表达丰度适中。内参和靶标基因miRNA在重复样本检测中一致性均较好(r>0.931,P<0.001)。根据3种软件分析结果,Cel-miR-39在70份脑脊液标本中表达稳定性更高,更适合作为脑脊液miRNA检测的内参基因。以Cel-miR-39为内参,靶标基因miR-126-3p(1.13±0.41比3.34±0.82,t=2.452,P=0.016)、miR-130a-3p(0.56±0.10比2.59±0.70,t=2.960,P=0.004)和miR-151a-3p(0.64±0.25比2.11±0.33,t=3.536,P=0.001)在结核性脑膜炎组脑脊液中的相对表达量均显著低于病毒性脑膜炎组。结论:Cel-miR-39可作为结核性脑膜炎患者脑脊液中miRNA定量检测的标准内参,脑脊液中miR-126-3p、miR-130a-3p和miR-151a-3p的相对表达量在结核性脑膜炎患者和病毒性脑膜炎患者之间存在差异。

Objective To explore the feasibility of U6 and Cel-miR-39 as reference genes for quantitative detection of microRNA(miRNA)in cerebrospinal fluid(CSF)of tuberculous meningitis(TBM),and validate the difference of miRNAs between tuberculous and viral meningitis(VM).Methods The remaining CSF specimens after routine examination were collected in Beijing Chest Hospital of Capital Medical University.A total of 36 TBM and 34 VM patients were enrolled based on the information in the medical records.Total RNA were extracted from the CSF samples,and Taqman based real-time quantitative PCR(RT-CR)analysis were performed to determine the concentration of the miRNAs in CSF.GeNorm,NormFinder and Bestkeeper software were used for stability analysis of the two reference genes.2-ΔCt method was used to determine the relative gene expression.Accordance of repeated tests was analyzed by Pearson correlation test.Continuous variables were compared by the t-test.Results Among the 70 samples,the average cycle threshold(Ct)value of U6 was 30.40±3.30,while the average Ct value of Cel-miR-39 was 21.49±0.70.The expression level of Cel-miR-39 was higher than that of U6.Correlation analysis showed good accordance of the repeated tests among the reference genes and target genes analysis in the randomly selected 10 samples(r>0.931,P<0.001).Based on the analyses results of the three software,including GeNorm,NormFinder and Bestkeeper,Cel-miR-39 presented better stability in RT-PCR analysis and was more suitable as a reference gene for miRNA quantitative determination in CSF sample of TBM patients.The relative expression levels of the three target miRNAs were calculated using Cel-miR-39 as the reference gene,and miR-126-3p(1.13±0.41 vs 3.34±0.82,t=2.452,P=0.016),miR-130a-3p(0.56±0.10 vs 2.59±0.70,t=2.960,P=0.004)and miR-151a-3p(0.64±0.25 vs 2.11±0.33,t=3.536,P<0.001)were showed significant lower expression levels in CSF in TBM group than that in VM group.Conclusions Cel-miR-39 can be used as a reference gene for quantitative detection of miRNAs in CSF of TBM patients.Significant differences were detected in expression level of miR-126-3p,miR-130a-3p and miR-151a-3p between TBM and VM group.