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1、1,生科4甲 蔡德峰,2,前言,sequencing of the human-functional genomicsGene-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 dur
2、ing normal development and in response to homeostatic challenges,4,Gene-expression microarrays,https:/www.vbi.vt.edu/article/articleview/145,5,RNA interferences(RNAi),www.mpg.de/./EEB/200432_035.shtml,(RNA-induced silencing complex),6,RNA interferences(RNAi),www.life.uiuc.edu/shapiro/RNAiApps.html,7
3、,pathfiles/m_atmPathway.asp,ATM/p53-mediatedATM/NFkB-mediated,G1 checkpoint,Ataxia-telangiectasia(AT),8,www.mbb.yale.edu/fl/fl_s_ghosh.htm,NFkB,ATM/NFkB-mediated,9,http:/,ATM/NFkB-mediated,NFkB,ATM,10,Hypothesis,the combined experimental strategy of expression arrays and RNAi is indeed a powerful me
4、thod 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,Microarrayanalysis,Database search,Computational promoter analysis,*-New candidate target genes,*,*,*,*,*,Ad
5、apted 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-downcellular systems,12,建立siRNA knocked-downcellular systems,Materials and methods,DNA fragments,To be transfec
6、ted,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 microarra
7、y hybridization,HEK293 cell,Materials and methods,(4 h with 200 ng/ml of NCS.),RNA,isolated using TRIzol reagenttreated with DNase Iphenol/chloroform extractedethanol-precipitated and quantitated.,Affymetrix Human Focus Gene-Chip arrays,(All samples were probed in independent triplicates),10 種狀態:fiv
8、e cellular systems(uninfected and the LacZ control cells and cellsknocked-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.RM
9、A 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 co
10、ntrols 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 treatment,17,Cluster analysis,Materials and methods,112 gene 使用 the EXPANDER package 去做 average-linkagehierarchical clustering,18,GO functional ge
11、ne 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,20,2
12、1,22,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/Jun,23,結論,RNAi,microarrays and computational promoter analysis 對於 dissection of transcriptiona
13、l 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 induction of much of the network,the two downstream regulators(p53/NFkB)mediated the activation of largely disjoint sets of genesStatistical tests 聯合 computational promoter analysis 是高精確的,
