【摘要】 背景:目前两种最可靠、应用最广的高通量RNA分离技术是基于异硫氰酸胍-酚︰氯仿的RNA分离技术和基于硅胶柱的RNA分离技术。在临床应用非常有限的组织材料时,有必要对RNA分离技术的可重复性和可靠性分别进行先期评价。目的:系统比较采用硅胶柱法和Trizol法从冻存、新鲜胃癌组织中分离总RNA的效果。方法:硅胶柱法和Trizol法分别提取长期-80℃冻存的(2年以上)和新鲜胃癌组织总RNA,紫外分光光度法比较RNA纯度,琼脂糖凝胶电泳分析28S和18S吸光度比值,进一步通过实时反转录-聚合酶链反应对扩增距mRNApolyA尾端不同大小片段的Ct值进行比较,间接判断不同大小mRNA的拷贝数及完整性。结果与结论:硅胶柱技术和Trizol方法在总RNA提取的纯度方面都表现很好。然而在RNA完整性方面,基于硅胶柱技术的提取方法要好于Trizol法,因为其核糖体RNA形式完好而且更好地保持了不仅短的及中等大小片段,对于较大的mRNA片段也保持更好。另外,与新鲜组织相比,长时间冻存对胃癌组织总RNA提取的影响很小。更多还原

【Abstract】 BACKGROUND:At present,the most reliable and commonly used methods are guanidine isothiocyanate based RNA isolation method(typically Trizol) and the silica-gel column based RNA isolation method.In case of clinical rare tissues,it is necessary to evaluate the reliability and repetitiveness of the optional RNA isolation methods.OBJECTIVE:To compare the silica-gel column based RNA extraction method and Trizol method to determine which technique is preferable when frozen,long-term stored or fresh gastric caricinoma tissues have to be evaluated for the downstream molecular analysis.METHODS:Frozen gastric carcinoma tissues were prepared by long-term storage(more than 2 years) in-80 ℃ while fresh tissues were harvested and processed immediately.The purity of RNA was determined with a spectrophotometer whereas the Ct values of target sequences were recorded by quantitative reverse transcription-PCR(qRT-PCR) system to indirectly assess the RNA intactness(28S and 18S) and mRNA fragment sizes.RESULTS AND CONCLUSION:The purity of RNA extracted with silica-gel column based and Trizol method was comparable.qRT-PCR data revealed that lower mean Ct values of 28 S and a longer fragment were obtained from RNA isolated with silica-gel column based method than Trizol method using fresh as well as frozen tissues.A low mean Ct value of 18 S and medium fragment were obtained in RNA isolated by silica-gel column based method from the fresh tissues,only.For the shorter fragment,no significant differences were observed.Silica-gel column based RNA isolation method was much more superior to typical Trizol method with respect to the reliable generation of an intact RNA and effective amplification of longer mRNA products in fresh as well as in frozen gastric carcinoma tissues.更多还原