不同预处理方法对提取尿液脱落细胞mRNA质量的影响  

Effects of Different Pretreatment Methods on mRNA Quality and Quantity of Exfoliated Urinary Cells

作　　者：王洁敏[1] 向芃[1] 顾乐怡[1] 

机构地区：[1]上海交通大学医学院附属仁济医院肾脏科

出　　处：《现代检验医学杂志》2016年第4期125-127,130,共4页Journal of Modern Laboratory Medicine

基　　金：国家自然科学基金资助项目(NO.81270781)

摘　　要：目的 探索合适的处理尿标本的方法获得尿脱落细胞mRNA。方法 比较尿液容量、尿液标本冻存时间、处理方式对提取尿液脱落细胞RNA的产量与质量的影响,并进行qPCR的试验。结果 40 ml尿液标本组及10 ml尿液标本组RNA总量均值分别为886.94±222.93 ng及211.22±62.01 ng,两组A260nm/A280nm比值分别为1.80±0.10和1.77±0.09; 尿液标本量与RNA总量相关（t=3.604,P=0.002）,不同尿液标本量RNA质量差异无统计学意义（t=0.708,P值〉0.05）。实时离心提取及冰冻保存尿液7天后提取两种预处理方法RNA总量分别为886.94±945.84 ng及881.50±829.02 ng,A260nm/A280nm比值分别为1.80±0.10及1.80±0.87; RNA总量与A260nm/A280nm比值差异无统计学意义（t=-0.221-0.085,P值均〉0.05）。直接冰冻尿液标本与TRIzol保存沉渣细胞两种预处理方法RNA总量分别为637.81±525.24 ng及639.86±535.42 ng,A260nm/A280nm比值分别为1.84±0.13及1.83±0.96; RNA总量与A260nm/A280nm比值差异无统计学意义（t=-0.47-0.293,P值均〉0.05）。尿脱落细胞mRNA中可以检测到足细胞的标记蛋白WT-1,podocin和synaptopodin的基因表达。结论 尿液标本量是提取脱落细胞RNA量的关键因素,-80℃低温冻存尿标本7天或Trizol预处理冻存均对提取尿液沉渣细胞RNA数量和质量无影响。Objective To explore a proper method to obtain exfoliated cells mRNA for detection in urine specimen. Methods Urine volume,different pretreating ways and frozen storage time on urinary sample were used to collect mRNA. A260nm/A280nm and A260nm/A280nm ratio,as well as qPCR were performed. Results Amount of mRNA was determinded by urine volume,but neither A260nm/A280nm or A260mm/A280nm ratio was realated with urine volume. Compared with real-time extraction process,freezing urine samples for 7 days showed no statistical difference （P〉0. 05） in total RNA and A260nm/A280nm or A260nm/A280nm ratio. There was no significant difference in total RNA and A260nm/A280nm or A260nm/A280nm ratio between direct freezing urine samples and Trizol-treated urine exfoliated cells. Podocyte marker including WT-1, podocin and synaptopodin mRNAs were detected in urine sediment cells. Conclusion Quantity of mRNA was determinded by urine volume. Directly freezing urine samples at -80℃ for 7 days or Trizol-treating urine sediment ceils have no effect on lowing quality and quantity of urinary sediment mRNA.

关 键 词：慢性肾脏病 尿液脱落细胞 MRNA 足细胞 

分 类 号：R446.12[医药卫生—诊断学]