摘要
目的 探讨Trizol法同时提取大鼠单侧海马组织RNA、基因组DNA和蛋白质的可行性及优势.方法Trizol法提取成年雄性Sprague-Dawley大鼠单侧海马组织RNA、基因组DNA以及蛋白质; 商业化试剂盒提取另一侧海马组织基因组DNA,RIPA裂解液提取蛋白质.用超微量分光光度计和SYBR实时定量RT-PCR法检测Trizol法提取的RNA的质量,利用超微量分光光度计和聚合酶链式反应（PCR）法比较两种方法提取DNA的效果及质量,采用Western blot法比较两种方法提取蛋白质的效果.结果 Trizol法提取单侧海马RNA的质量浓度介于1178.3～2225.8ng/μL之间,A260/280介于2.01～2.03,A260/230介于1.88～2.22,并获得了稳定的β-actin和GR循环域值.Trizol法和试剂盒提取单侧海马DNA的质量浓度分别介于215.7～271.8ng/μL和268.6～872.5ng/μL之间,均适于充当PCR反应模板.Trizol法和RIPA裂解液提取的蛋白质在Western blot法检测目的 蛋白含量的过程中均呈现清晰的条带.结论 Trizol法提取大鼠单侧海马组织RNA的同时提取的DNA和蛋白质,与商业化试剂盒和RIPA裂解液从不同海马组织中分别提取基因组DNA和蛋白质的效果相当.采用Trizol法同时提取海马组织的RNA、DNA和蛋白质,不仅提高了实验效率、节约了实验成本,而且有效避免了因反复实验操作带来的人为干扰及误差.
Objective To explore the feasibility and advantages of the Trizol reagent method of extracting total RNA, genomic DNA and protein from rat hippocampus. Methods RNA, DNA and protein were extracted with Trizol method from one side of the hippocampus of adult male Sprague-Dawley rats. Genomic DNA and protein were extracted by commercial kit and RIPA buffer lysis, respectively, from the other side of the hippocampus. We measured the quality of RNA by spectrophotometer and RT-PCR, the quality of DNA by spectrophotometer and PCR, and the quality of protein by Western blot. Results The concentration of the total RNA extracted by Trizol reagent was 1 178.3 ng/μL--2 225.8 ng/μL, A260/280 ratio was 2.01 -- 2.03, and A260/230 ratio was 1.88-- 2.22. The cycle thresholds of t3-actin and GR were stable. The concentration of the DNA extracted by Trizol reagent and commercial kit was 215.7 ng/μL-- 271.8 ng/μL and 268.6 ng/taL-- 872.5ng/taL, respectively. There were clear bands of target proteins in Western blot utilizing proteins extracted by Trizol reagent and RIPA buffer lysis. Conclusion The effects of extracting DNA and protein from hippocampal tissue using Trizol reagent, commercial kit and RIPA buffer lysis are similar. Therefore, the method of extracting total RNA, DNA and protein by Trizol reagent can improve efficiency, save experiment cost and reduce unnecessary interference and errors in the experi- ment.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第2期276-280,共5页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81200845)~~
