已阅读5页,还剩18页未读, 继续免费阅读
版权说明:本文档由用户提供并上传,收益归属内容提供方,若内容存在侵权,请进行举报或认领
文档简介
1,生科4甲 蔡德峰,2,前言,sequencing of the human - functional genomics Gene-expression microarrays and RNA interferences (RNAi) ATM/NFB and ATM/p53-mediated arms,3,functional genomics,to gaining system-level understanding of the mechanisms gene products interact and regulate each other physiological processes during normal development and in response to homeostatic challenges,4,Gene-expression microarrays,/ article/articleview/145,5,RNA interferences (RNAi),www.mpg.de/./ EEB/200432_035.shtml,(RNA-induced silencing complex),6,RNA interferences (RNAi),/ shapiro/RNAiApps.html,7,/ pathfiles/m_atmPathway.asp,ATM/p53 -mediated ATM/NFkB -mediated,G1 checkpoint,Ataxia- telangiectasia ( AT),8,/ fl/fl_s_ghosh.htm,NFkB,ATM/NFkB -mediated,9,/popup/cbc/NFKB_Interactive_Pathway.htm,ATM/NFkB -mediated,NFkB,ATM,10,Hypothesis,the combined experimental strategy of expression arrays and RNAi is indeed a powerful method for the dissection of complex transcriptional networks, and that computational promoter analysis can provide a strong complementary means for assessing the accuracy of this dissection.,11,Microarray analysis,Database search,Computational promoter analysis,*- New candidate target genes,*,*,*,*,*,Adapted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2001),TRANSFAC,實驗流程圖,Definition of the damage-responding gene set,Cluster analysis,GO functional gene annotations,建立siRNA knocked-down cellular systems,12,建立siRNA knocked-down cellular systems,Materials and methods,DNA fragments,To be transfected,To be cloned,pSUPER retroviral vector,HEK293 cell (哺乳動物),(selected with puromycin or hygromycin),病毒載體用于siRNA表達,其優勢在于可以直接高效率感染細胞進行基因沉默的研究,避免由于質粒轉染效率低而帶來的種種不便,而且轉染效果更加穩定。 最適用于:已知一個有效的siRNA序列,需要維持較長時間的基因沉默。,13,以Western blotting 檢驗 RNAi,RNAi并不能完全阻斷基因的表達,特別是表達異常高的基因。,14,Sample preparation and microarray hybridization,HEK293 cell,Materials and methods,(4 h with 200 ng/ml of NCS.),RNA,isolated using TRIzol reagent treated with DNase I phenol/chloroform extracted ethanol-precipitated and quantitated.,Affymetrix Human Focus Gene- Chip arrays,(All samples were probed in independent triplicates),10 種狀態 : five cellular systems (uninfected and the LacZ control cells and cells knocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatment and 4 h after exposure to NCS.,15,Computation of gene expression levels from microarray signals,Materials and methods,RMA method,1. RMA 計算後, 信號明顯增強 2. RMA 使用齊次多項式證明數據改進更好,16,Definition of the damage-responding gene set,Materials and methods,DMA method 取數值at least 1.5-fold in one control (either the uninfected or the LacZ-infected cells), and at least 1.4-fold in the same direction in the other control.,A total of 112 genes that were induced in both controls met this criterion and are referred to as the damage-induced gene set. Only seven genes met an analogous criterion for repression in response to NCS treatment,17,Cluster analysis,Materials and methods,112 gene 使用 the EXPANDER package 去做 average-linkage hierarchical clustering,18,GO functional gene annotations,Materials and methods,The gene ontology (GO) annotations,Computational promoter analysis,Materials and methods,PRIMA software,19,Quantitative real-time RT-PCR,Materials and methods,Five micrograms of total RNA,cDNA,oligo(dT) SuperScript II RNase H- reverse transcriptase,real-time PCR,20,21,22,討論,RNAi and microarray technologies and a recently developed computational tool are powerful off-target effects computational promoter analysis was highly enriched for the binding signature of ATF2/ATF3/Jun,23,結論,RNAi, microarrays and computational promoter analysis 對於 dissection of transcriptional networks 的研究是有力的 Targeting the primary activator of a DNA damage response network, the upstream regulator(ATM) was indeed requi
温馨提示
- 1. 本站所有资源如无特殊说明,都需要本地电脑安装OFFICE2007和PDF阅读器。图纸软件为CAD,CAXA,PROE,UG,SolidWorks等.压缩文件请下载最新的WinRAR软件解压。
- 2. 本站的文档不包含任何第三方提供的附件图纸等,如果需要附件,请联系上传者。文件的所有权益归上传用户所有。
- 3. 本站RAR压缩包中若带图纸,网页内容里面会有图纸预览,若没有图纸预览就没有图纸。
- 4. 未经权益所有人同意不得将文件中的内容挪作商业或盈利用途。
- 5. 人人文库网仅提供信息存储空间,仅对用户上传内容的表现方式做保护处理,对用户上传分享的文档内容本身不做任何修改或编辑,并不能对任何下载内容负责。
- 6. 下载文件中如有侵权或不适当内容,请与我们联系,我们立即纠正。
- 7. 本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。
最新文档
- 湖南省湘潭市2024届中考联考物理试卷含解析
- 2023-2024学年河北省唐山市友谊中学中考一模物理试题含解析
- 2024届江苏省兴化顾庄学区七校联考中考物理考试模拟冲刺卷含解析
- 泰安市重点名校2024届中考物理考试模拟冲刺卷含解析
- 新疆吐鲁番市高昌区第二中学2022年数学高三上期末统考试题含解析
- 天津市五校2022年数学高三第一学期期末学业水平测试试题含解析
- 上海市重点名校2022-2023学年数学高三第一学期期末调研模拟试题含解析
- 陕西省咸阳市礼泉县2022-2023学年数学高三第一学期期末综合测试模拟试题含解析
- 云南省玉溪市名校2024年中考物理押题试卷含解析
- 国际体育门卫工作合同范本
- 北京市石景山区2022-2023学年七年级下学期期末地理试题
- 江苏省苏州区六校联考2023-2024学年中考联考英语试题含答案
- 期末检测(试题)-2023-2024学年四年级下册数学人教版
- 2024年上海市中考语文真题(无答案)
- 统编版2023-2024学年高二语文下学期期末模拟卷1(有详解)
- JTG-D40-2011公路水泥混凝土路面设计规范
- 芯片资源开发方案
- XX学校物业项目投标文件
- 中国农业文化遗产与生态智慧智慧树知到期末考试答案章节答案2024年浙江农林大学
- 水平二田径大单元
- 部编版四年级语文下册段落句子病句修改
评论
0/150
提交评论