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LncRNA表达载体的构建
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NCBI和NONCODE数据库可以找到全长序列的lncRNA，还需要做RACE吗？
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请教：通过测序或者芯片结果找到的lncRNA，如果这个lncRNA在NCBI和NONCODE数据库可以找到全长序列的lncRNA，还需要做RACE吗？谢谢！
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关于丁香园上传用户：nxyzunlofh资料价格：5财富值&&『』文档下载 ：『』&&『』学位专业：&关 键 词 ：&&&&权力声明：若本站收录的文献无意侵犯了您的著作版权，请点击。摘要:（摘要内容经过系统自动伪原创处理以避免复制，下载原文正常，内容请直接查看目录。）非编码RNA(noncoding RNA, ncRNA)是最近几年来发明的一类在生物体内广泛存在并在性命运动中行使主要功效的RNA份子，与mRNA、tRNA和rRNA分歧，其不编码卵白质，也不直接介入卵白质的分解。NcRNA普遍存在于从高等到高级的多种生物中，品种单一，重要包含：miRNA(microRNA)、piRNA、gRNA、lncRNA (long noncoding RNA)等。LncRNA是一类长度年夜于200个碱基，缺乏完全的开放浏览框，无或很少有卵白编码才能的RNA。研讨注解lncRNA在细胞心理和病理运动中施展主要的感化，并介入包含肿瘤在内的多种疾病的产生成长进程。本文研讨了lncRNA异常表达与肝细胞癌(Hepatocellular Carcinoma, HCC)关系。应用基因表达谱测序法和及时定量PCR检测了lncRNA在肝癌和癌旁组织间的表达，发明GAS5、NCRNA00107、NCRNA00115、SNHG6和MEG3等lncRNA在两种组织间存在明显性的差别表达。经由过程构建lncRNA真核表达载体，并在癌细胞中过表达，应用及时定量PCR、荧光素酶申报基因、免疫印迹、表达谱芯片、RNA-pulldown和RIP等技巧，发明MEG3经由过程与p53卵白互相感化延伸p53卵白的半衰期，增进p53的转录活性，进而克制肝癌细胞增殖，增进肝癌细胞凋亡。本研讨还经由过程Arraystar Human LncRNA Microarray V2.0芯片对肝细胞癌组织和癌旁组织中一切差别表达的lncRNA和mRNA停止了年夜范围剖析，取得了肝癌相干lncRNA，并构建了lncRNA-mRNA共表达收集，为深刻研讨肝癌产生成长进程中lncRNA的感化机制供给了基本。获得的重要停顿有：1.经由过程基因表达谱测序剖析，发明肝癌组织中部门lncRNA异常表达，定量PCR成果显示GAS5、NCRNA00107、NCRNA00115和SNHG6在肝细胞癌组织中表达降低，而MEG3表达明显降低。2. MEG3具有克制肝癌细胞增殖及克隆构成的才能，在肝癌细胞HepG2细胞中过表达MEG3可以明显地增进细胞凋亡。3. MEG3可调控p53的转录活性及其靶基因的表达。过表达MEG3可招致p53卵白程度增长，并增进p53的转录活性。4. RNA pulldown和RIP试验证实MEG3与p53直接互相感化，并发明p53经由过程其DNA联合构造域与MEG3联合。MEG3截短体的荧光素酶试验成果证实全长MEG3关于其增进p53转录活性的感化是必须的。5.经由过程lncRNA芯片及统计剖析发明有214条lncRNA和338条mRNA与肝癌相干。针对338条mRNA停止GO剖析，成果显示富集最明显的生物学进程包含细胞代谢进程、细胞构成成份组装、细胞周期进程和对化学安慰物的反响进程。6.在3对肝癌和癌旁样本中采取及时定量PCR验证了芯片中50条差别表达的lncRNA，成果显示40/50的lncRNA表达趋向与芯片成果是分歧的。在19对肝癌和癌旁样本中经由过程及时定量PCR检测了从芯片成果中挑选的8个lncRNA，成果显示这些lncRNA的表达变更与芯片成果根本分歧。同时，还发明这些lncRNA及其周边编码卵白基因在肝癌及癌旁组织的表达变更趋向根本分歧。7.构建了肝癌相干lncRNA-mRNA共表达收集。采取生物信息学剖析3例肝癌和癌旁组织的lncRNA芯片的成果，在全基因组规模内寻觅在肝癌产生成长进程中差别表达的lncRNA和mRNA。依据其表达变更差别，构建了lncRNA-mRNA共表达收集。综上所述，本论文提醒了在肝癌中异常表达的lncRNAMEG3能联合p53卵白，影响其半衰期和转录活性，进而克制肝癌细胞增殖，增进细胞凋亡。同时，应用lncRNA芯片及生物信息学剖析，发明了一批肝癌相干lncRNA，并构建了肝癌相干lncRNA-mRNA共表达收集，为从全基因组规模内研讨肝癌病发机制供给了必定的基本。Abstract:Non coding RNAs noncoding RNA (ncRNA) is in recent years to invent a kind of in vivo widespread and the main function of RNA molecules in the midst of the life movement, and mRNA, tRNA and rRNA differences, its coding protein, not directly involved in the decomposition of protein. NcRNA generally exists in a variety of biological high until the advanced, single species, include: miRNA (microRNA), piRNA, gRNA, lncRNA (long noncoding RNA). Lncrna is the length of a class of the eve of the 200 bases, the lack of open completely browsing box, no or little protein coding to RNA. Research shows the lncRNA in the cell and pathological psychological movement play important role in many diseases and tumors, which contains in the process of growth. This paper studies the abnormal expression of lncRNA and hepatocellular carcinoma (Hepatocellular, Carcinoma, HCC) relationship. Application of gene expression profiling to sequencing and real-time quantitative PCR to detect the lncrna expression in hepatocellular carcinoma and adjacent tissues, invention gas5, NCRNA00107, NCRNA00115, SNHG6 and meg3 lncrna between the two organizations exist obvious difference expression. Through the process of building a lncrna eukaryotic expression vector and in cancer cells overexpressing and application in quantitative PCR and luciferase reporting gene, Western blot, expression profiling, RNA-pulldown and rip techniques, the invention meg3 through and p53 protein affects mutually extends the half-life of the p53 protein, enhance the transcriptional activity of p53 and inhibit proliferation of hepatoma cells, enhance the apoptosis of hepatoma cells. This study also through process Arraystar human lncrna microarray v2.0 chip of lncrna all the difference expression in HCC tissues and adjacent tissues and mRNA stop the large-scale analysis and achieved lncrna HCC coherent, and construct lncRNA-mRNA co expression network, deep research discussion tumor growth in the process of lncrna action mechanism that can provide the basis. Have obtained important pause: 1. Via gene expression profile sequencing analysis, the invention in hepatocellular carcinoma (HCC), Department of lncrna abnormal expression and quantitative PCR results showed gas5, NCRNA00107, NCRNA00115 and SNHG6 in hepatocellular carcinoma tissue reduced expression and meg3 expression decreased obviously. 2 MEG3 with hepatocellular carcinoma cell proliferation and clone restraint which can, in HepG2 cells overexpressing MEG3 can significantly promote cell apoptosis. 3 expression of MEG3 transcriptional activity and target gene regulation of p53. Overexpression of MEG3 can lead to p53 protein levels of growth, and enhance the transcription activity of p53. 4 RNA pulldown and RIP test confirmed that MEG3 and p53 directly interact with each other, and the invention of p53 through its DNA domain and MEG3 combined structure. Luciferase test results of truncated MEG3 confirmed the full-length MEG3 is a must on the enhance the transcriptional activity of p53 effect. 5 through lncRNA chip and statistical analysis of invention are 214 lncRNA and 338 mRNA of hepatocellular carcinoma and coherence. In 338 mRNA GO analysis, the results showed the most obvious enrichment of biological processes including cell metabolism process, cell component assembly, cell cycle progression and to comfort the chemical reaction process. 6 in 3 pairs of HCC and adjacent liver samples by quantitative real-time PCR to verify the expression of 50 different chip lncRNA, results show the expression of lncRNA and 40/50 results are consistent with the trend of the chip. In 19 pairs of HCC and adjacent liver samples through timely and quantitative detection of PCR lncRNA in 8 selected from the chip results, the results showed that the expression of change and results of fundamental differences between these lncRNA chips. At the same time, the invention of the lncrna and its surrounding encoding protein gene expression in hepatocellular carcinoma and paracancerous tissue change trend of fundamental differences. 7 constructed HCC coherent lncRNA-mRNA co expression collection. By bioinformatics analysis of 3 cases of hepatocellular carcinoma lncRNA chip results on a genome-wide scale for the expression difference between the growth process of lncRNA and mRNA in hepatocellular carcinoma. On the basis of the change of expression difference, constructs a collection of co expression of lncRNA-mRNA. In summary, this paper reminded lncRNAMEG3 in hepatocellular carcinoma (HCC) abnormal expression of p53 protein, affecting its half-life and transcriptional activity, and inhibit proliferation of hepatoma cells, promote cell apoptosis. At the same time, application lncrna chip and bioinformatics analysis, invented a number of hepatocellular carcinoma (HCC) coherent lncrna and construct the hepatocellular carcinoma (HCC) coherent lncRNA-mRNA co expression of the gathering, as from a genome-wide scale research of liver cancer mechanism provides certain basic.目录:中文摘要4-6ABSTRACT6-8英文缩写注释表9-15第一章 绪论15-27&&&&1.1 长链非编码 RNA 概述15-18&&&&1.2 LncRNA 的生物学功能18-21&&&&&&&&1.2.1 表观遗传学调控18-19&&&&&&&&1.2.2 转录调控19-20&&&&&&&&1.2.3 转录后调控20-21&&&&1.3 LncRNA 与疾病21-23&&&&&&&&1.3.1 LncRNA 与肿瘤21-22&&&&&&&&1.3.2 LncRNA 与神经退行性疾病22-23&&&&1.4 LncRNA 研究策略23-25&&&&&&&&1.4.1 高通量分析 lncRNA 表达23-24&&&&&&&&1.4.2 海量数据分析和鉴定24&&&&&&&&1.4.3 LncRNA 和蛋白相互作用研究方法24-25&&&&1.5 MEG3 的发现、特征及功能25-27第二章 LncRNAMEG3 在肝癌细胞中的功能及作用机制研究27-67&&&&2.1 实验材料27-32&&&&&&&&2.1.1 组织标本27&&&&&&&&2.1.2 菌株、细胞系及质粒27-28&&&&&&&&2.1.3 主要仪器28&&&&&&&&2.1.4 主要试剂与材料28-29&&&&&&&&2.1.5 主要溶液的配制29-32&&&&&&&&2.1.6 引物和 siRNA 序列32&&&&2.2 实验方法32-46&&&&&&&&2.2.1 细胞培养32-33&&&&&&&&2.2.2 总 RNA 的提取和实时定量 PCR33-35&&&&&&&&2.2.3 构建 MEG3 表达载体35-37&&&&&&&&2.2.4 细胞转染37-38&&&&&&&&2.2.5 MEG3 稳定株的筛选38-39&&&&&&&&2.2.6 CCK8 检测细胞增殖和克隆形成实验39&&&&&&&&2.2.7 细胞周期和凋亡检测39-40&&&&&&&&2.2.8 Western blot40-42&&&&&&&&2.2.9 双荧光素酶报告基因实验42&&&&&&&&2.2.10 RNA pulldown 和 RNA 免疫共沉淀42-46&&&&&&&&2.2.11 p53 蛋白半衰期检测46&&&&2.3 实验结果46-62&&&&&&&&2.3.1 基因表达谱测序筛选肝癌组织中差异表达 ncRNA46-47&&&&&&&&2.3.2 部分 lncRNA 在肝癌组织中表达失调47-48&&&&&&&&2.3.3 MEG3、GAS5、SNHG6 和 NCRNA00107 表达载体构建48-49&&&&&&&&2.3.4 MEG3 抑制肝癌细胞增殖49-50&&&&&&&&2.3.5 MEG3 促进肝癌细胞凋亡50-52&&&&&&&&2.3.6 MEG3 增强 p53 转录活性52-54&&&&&&&&2.3.7 MEG3 与 p53 相互作用54-56&&&&&&&&2.3.8 MEG3 调控 p53 下游靶基因56-62&&&&2.4 讨论和总结62-67&&&&&&&&2.4.1 讨论62-65&&&&&&&&2.4.2 总结65-67第三章 基于芯片分析肝癌中的 lncRNA 表达模式67-83&&&&3.1 实验材料67-68&&&&&&&&3.1.1 病人肿瘤样本67-68&&&&&&&&3.1.2 主要试剂与材料68&&&&&&&&3.1.3 主要仪器68&&&&&&&&3.1.4 引物68&&&&3.2 实验方法68-69&&&&&&&&3.2.1 组织总 RNA 提取和实时定量 PCR68&&&&&&&&3.2.2 芯片分析和生物信息学分析68-69&&&&3.3 实验结果69-79&&&&&&&&3.3.1 肝癌和癌旁组织 RNA 鉴定69-70&&&&&&&&3.3.2 肝癌组织中 lncRNA 和 mRNA 的表达谱70-71&&&&&&&&3.3.3 LncRNA 分类和亚类分析71&&&&&&&&3.3.4 差异表达 mRNA 的功能富集71-72&&&&&&&&3.3.5 差异表达 lncRNA 的基因组位置72-73&&&&&&&&3.3.6 LncRNA-mRNA 共表达网络73-76&&&&&&&&3.3.7 部分 lncRNA 在肝癌组织中的表达失调76-79&&&&3.4 讨论和总结79-83&&&&&&&&3.4.1 讨论79-81&&&&&&&&3.4.2 总结81-83参考文献83-95附录95-101个人简历101-103致谢103-104分享到：相关文献|}