生科4甲蔡德峰_第1页
生科4甲蔡德峰_第2页
生科4甲蔡德峰_第3页
生科4甲蔡德峰_第4页
生科4甲蔡德峰_第5页
已阅读5页,还剩18页未读 继续免费阅读

下载本文档

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1前言sequencing of the human - functional genomicsGene-expression microarrays and RNA interferences (RNAi)ATM/NFB and ATM/p53-mediated arms2functional genomicsto gaining system-level understanding of the mechanisms gene products interact and regulate each other physiological processes during n

2、ormal development and in response to homeostatic challenges3Gene-expression microarrays article/articleview/1454RNA interferences (RNAi) EEB/200432_035.shtml(RNA-induced silencing complex)5RNA interferences (RNAi) shapiro/RNAiApps.html6 pathATM/p53 -mediatedATM/NFkB -mediatedG1 checkpointAtaxia- tel

3、angiectasia ( AT)7 fl/fl_s_ghosh.htmNFkBATM/NFkB -mediated8ATM/NFkB -mediatedNFkBATM9Hypothesisthe 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 s

4、trong complementary means for assessing the accuracy of this dissection.10MicroarrayanalysisDatabase searchComputational 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 setCluste

5、r analysisGO functional gene annotations建立siRNA knocked-downcellular systems11建立siRNA knocked-downcellular systemsMaterials and methodsDNA fragmentsTo be transfectedTo be clonedpSUPER retroviral vectorHEK293 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

6、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。12以Western blotting 檢驗 RNAiRNAi并不能完全阻斷基因的表達,特別是表達異常高的基因。13Sample preparation and microarray hybridizationHEK293 cellMaterials and methods(4 h with 200 ng/ml of NCS.)RNAisolated using TRIzol reagenttreated with DNase Iphenol/chloroform ex

7、tractedethanol-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 cellsknocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatmen

8、t and 4 h after exposure to NCS.14Computation of gene expression levels from microarray signalsMaterials and methodsRMA method1. 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15Definition of the damage-responding gene setMaterials and methodsDMA method 取數值at least 1.5-fold in one control (either the uninfect

9、ed 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 thiscriterion and are referred to as the damage-induced gene set.Only seven genes met an analogous criterion for repression in response to NCS

10、treatment16Cluster analysisMaterials and methods112 gene 使用 the EXPANDER package 去做 average-linkagehierarchical clustering17GO functional gene annotationsMaterials and methodsThe gene ontology (GO) annotationsComputational promoter analysisMaterials and methodsPRIMA software18Quantitative real-time

11、RT-PCRMaterials and methodsFive micrograms of total RNAcDNA oligo(dT) SuperScript II RNase H- reverse transcriptasereal-time PCR192021討論RNAi and microarray technologies and a recently developed computational tool are powerfuloff-target effectscomputational promoter analysis was highly enriched for the binding signature of ATF2/ATF3/Jun22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 required for the indu

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。

评论

0/150

提交评论