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AIM To isolate, done and sequence gcys-18 overexpressed in gastric carcinoma.METHODS gcys-18 was isolated from differential display gel between GC7901 and GES-1 by mRNAdifferential display PCR, and was cloned into T vector. As a probe gcys-18 was hybridized to total RNAs ofGC7901 and GES-l, and was sequenced. Its sequence was screened against GeneBank. According to theobtained sequence, a pair of primers were designed and used to examine 26 specimens of gastric cancers andcorresponding paracancerous tissues by quantitative reverse transcriptase PCR.RESULTS gcys-18 was isolated and cloned, and confirmed to be expressed higher in GC7901 than in GES-1 by RNA dot blot; gcys-18 was 416bp, and partly similar to HEK5, and its accepted number in GeneBankwas AF071057; 18 out of 26 specimens of gastric cancers and 2 out of corresponding paracancerous tissueswere examined by RT-PCR.CONCLUSION gcys-18 may be an important expressed sequence tag in gastric cancer, and takes part inprogression of gastric carcinoma.
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RNA快速提取一步法的建立
RNA是分子生物学研究的一个重要组成部分, 不论是cDNA文库的构建、RT-PCR、RNA序列分析、差异显示(mRNA differential display)、代表性差异分析 (representational Difference Analysis, RDA)、抑制性消减杂交(suppression Subtraction Hybridization, SSH)、Northern印迹分析、蛋白质的体外翻译等均依赖于高纯度完整的RNA. 因此, 如何获得高质量的RNA是研究人员关注的问题.1987年Chomczynski等[1]建立了酸-异硫氰酸胍-苯酚-氯仿一步法(acid-guanidine-phenol-chloroform, AGPC).该方法较以往的传统方法操作简单,无需特殊的设备,抽提时间缩短至4 h,因而得到广泛的应用.数家公司据此开发出RNA提取试剂盒[2,3].使用试剂盒简单方便,但价格相对昂贵.为探索操作简便、经济实用、快速有效的RNA提取方法,我们分析了大量相关文献资料,进行了多次实验,成功地研制出一种新型的RNA提取试剂,建立了其操作程序,我们称之为RNA快速提取一步法.其性能达到进口试剂盒的水平,但造价低廉、操作简便,特别适用于国内实验室.
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几种基因差异表达分析方法的比较
高等生物大约含有100 000个不同的基因,其中仅有约15%得到表达,基因表达的变化是调控细胞生命活动过程的核心机制[1],因此,分析基因表达的差异不仅在发育、分化和突变等研究领域有着极大的应用价值,而且已成为基因克隆的有效手段之一,其技术发展也非常迅速.目前主要有差减杂交(subtractive hybridization,SH)、mRNA差异显示逆转录PCR(mRNA differential display reverse transcription PCR,DDRT-PCR)、cDNA代表性差异分析(cDNA representational difference analysis,cDNARDA)和抑制消减杂交(suppression sbtractive hybridization,SSH)等.尽管这些具有代表性的方法均不完善,不能通过它们得到所有的差异表达基因,但已经有大量的重要基因通过这些方法得到了分离和鉴定.本文就这些技术的主要原理、基本过程、优越性和主要缺陷做一比较分析.
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差异显示PCR分析高脂饮食诱导的肥胖大鼠脂肪组织的基因表达谱
本实验采用高脂饮食饲养SD大鼠,建立肥胖大鼠模型,用差异显示PCR(DD-PCR)方法比较高脂饲料组大鼠与普通饲料组大鼠脂肪组织中基因表达的差异,力求发现受饮食调控的基因,探讨高脂饮食致肥胖的分子机制.一、材料和方法1.动物模型与标本采集:20只SD大鼠分为高脂饲料组和普通饲料组.动物饲养16周.2.脂肪组织总RNA的抽提及处理:(1)脂肪组织总RNA的抽提:取1 g大网膜脂肪组织按照Trizol总RNA抽提试剂盒说明书进行.(2)总RNA的处理:用不含RNA酶的DNA酶处理抽提的总RNA以去除含有的少量基因组DNA.