改良的Trizol法提取小鼠皮肤RNA技术  

Modified Trizol Method for Extracting RNA from Mouse Skin

作　　者：林震嘉 张辉[2] 揭英桃 熊媖 唐源 谈智[1] 刘先国[1] 周利君[1] LIN Zhen-jia;ZHANG Hui;JIE Ying-tao;XIONG Ying;TANG Yuan;TAN Zhi;LIU Xianguo;ZHOU Li-jun(Department of Physiology,Zhongshan School of Medical,Sun Yat-sen University//Center for Pain Research,Sun Yat-sen University,Guangzhou 510080,China;Department of Anesthesiology,Guangdong Second People′s Hospital,Guangzhou 510317,China)

机构地区：[1]中山大学中山医学院生理教研室//疼痛研究中心,广东广州510080 [2]广东省第二人民医院麻醉科,广东广州510317

出　　处：《中山大学学报（医学科学版）》2020年第2期224-232,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81771204,81270377);广东省自然科学基金(2019A1515011838)。

摘　　要：[目的]由于皮肤组织韧性强且存在大量RNA酶,采用经典Trizol法提取的RNA存在易降解质量低等不足.因此,本研究旨在通过改良的Trizol法实现高质量的皮肤RNA提取.[方法]采用不同处理方式(Tri:Trizol包埋;Pro:RNA样本保护液包埋;Cry:先冷冻液氮再冷冻-80℃冰箱;LNG:液氮研磨;Cut:采用剪刀剪碎组织)提取的小鼠正常皮肤RNA为实验组;脊髓组织作为参照组,并以咪喹莫特(IMQ)诱导的小鼠银屑病模型皮肤组织作为保真性验证组.我们采用紫外分光光度法、琼脂凝胶电泳法、定量逆转录PCR法(qRT-PCR)等方法来检测经典Trizol法(1-Tri,Nor)和改良Trizol法(2-Tri,LNG-Tri,Tri-Cut,Pro)提取的RNA浓度、纯度和完整性以及IL-1βmRNA表达的差异性情况.[结果]①与脊髓组织相比,经典Trizol法(1-Tri)获得的正常皮肤组织总RNA量要低,DNA污染和5S RNA条带明显,IL-1βmRNA相对表达偏高,说明经典Trizol法在皮肤组织中提取RNA存在局限性,需要进行改良.②在不同小鼠皮肤样本处理组中,改良的2-Tri法和LNG-Tri法获得的RNA浓度更高,同时RNA降解、DNA污染更低,IL-1βmRNA表达更接近正常水平.更重要的是,在小鼠银屑病模型中,2-Tri法提取的RNA样本能真实反映出正常皮肤与银屑病变皮肤之间IL-1βmRNA的变化趋势.[结论]改良的2-Tri或LNG-Tri法分离的总RNA质量好,能可靠地反映生理或病理条件下的mRNA表达模式.【Objective】Due to the tough nature of skin tissue and a high presence of RNases,the isolation of skin RNA by the classical Trizol method presents a challenge.Therefore,we adapted and tested different sample treatment protocols to improve the Trizol method for high-quality extraction of skin RNA.【Methods】In this study,normal skin of mice processed by different treatments(Tri:submersion Trizol;Pro:RNA sample protector;Cry:cryopreservation in liquid nitrogen frozen and then-80℃refrigerator;LNG:liquid nitrogen grinding;Cut:scissor cutting)were used as the experimental groups.Spinal cord tissue(Sp)was used as the reference group,and skin tissue of mouse psoriasis model induced by imiquimod(IMQ)was used as the validation group.We compared skin RNA concentration,purity and integrity,as well as IL-1βmRNA expression extracted by conventional Trizol methods(1-Tri,Nor)and modified Trizol methods(2-Tri,LNG-Tri,Tri-Cut,Pro),which were determined by UV spectrophotometry,agar gel electrophoresis and quantitative reverse transcription PCR(qRT-PCR).【Results】①Compared with spinal cord(Sp),the total RNA of normal skin tissue extracted with the same classical Trizol method(1-Tri)was with lower yields,more obvious DNA contamination and 5S RNA bands,and higher IL-1βmRNA relative expression,suggesting that skin tissue was relatively special and the classical Trizol methods of skin RNA extraction should be improved.②Among the different treatment methods of skin tissue,2-Tri and LNG-Tri methods resulted in higher RNA concentrations,lower RNA degradation and lower DNA contamination,and the expression of IL-1βmRNA was closer to normal levels.More importantly,the skin RNA samples extracted by the 2-Tri method can reflect more realistically the variation of IL-1βmRNA expression between normal and psoriasiform groups.【Conclusion】Improved 2-Tri or LNG-Tri method has the advantage of high quality of total RNA,and 2-Tri can more reliably reflect the mRNA expression pattern under physiological and pathological conditions

关 键 词：RNA提取 小鼠皮肤 TRIZOL 银屑病 

分 类 号：Q5-3[生物学—生物化学]