转录组学transcriptomicswxj.ppt
《转录组学transcriptomicswxj.ppt》由会员分享,可在线阅读,更多相关《转录组学transcriptomicswxj.ppt(55页珍藏版)》请在三一文库上搜索。
1、Transcriptomics,转录组学,Transcriptome: An evolving definition,(The population of) mRNAs expressed by a genome at any given time (Abbott, 1999) The complete collection of transcribed elements of the genome. (Affymetrix, 2004),Transcribed elements,mRNAs: 35, 913 transcripts (including alternative spliced
2、 variants) Non-coding RNAs tRNAs (497 genes) rRNAs (243 genes) snmRNAs (small non-messenger RNAs) microRNAs and siRNAs (small interferring RNAs) snoRNAs (small nucleolar RNAs) snRNAs (small nuclear RNAs) Pseudogenes ( 2,000),Transcriptomics,Definition The study of characteristics and regulation of t
3、he functional RNA transcript population of a cell/s or organism at a specific time. Scope the population of functional RNA transcripts. the mechanisms that regulate the production of RNA transcripts dynamics of the trancriptome (time, cell type, genotype, external stimuli),一、转录组学研究全部RNA的表达及功能,转录组(tr
4、anscriptome)指特定状态下一种细胞或组织所能转录出来的所有RNA的总和。包括编码RNA,即mRNA和非编码RNA (non-coding RNA, ncRNA) 转录组学(transcriptomics):是在整体水平上研究细胞基因转录情况及转录调控规律的科学。 RNA组学(RNomics):是分析、鉴定非信使小RNA(small non-messenger RNA, snmRNA)在特定状态下表达情况、功能及其与蛋白质的相互作用。,转录组的特点:受到内外多种因素的调节,因而是动态可变的。能够揭示不同物种、不同个体、不同细胞、不同发育阶段及不同生理病理状态下的基因差异表达信息。,Ob
5、serving the transcriptome,Focussed Experimental Approaches: Northern Blotting Analysis RT-PCR (quantitative or semi-quantitative) High throughput Approaches: Closed System Profiling: Microarray expression profiling Open System Profiling: Serial analysis of gene expression (SAGE) Massively Parallel S
6、ignature Sequencing (MPSS),微阵列(microarray) SAGE MPSS,研究技术,(一)微阵列是大规模基因组表达谱研究的主要技术,大规模表达谱或全景式表达谱(global expression profile):是生物体(组织、细胞)在某一状态下基因表达的整体状况。 微阵列或基因芯片(DNA chip):利用光导化学合成、照相平板印刷以及固相表面化学合成等技术,在固相表面合成成千上万个寡核苷酸探针,并与放射性同位素或荧光物标记的来自不同细胞、组织或整个器官的DNA或mRNA反转录生成的第一链cDNA进行杂交,然后用特殊的检测系统对每个杂交点进行定量分析。,Ex
7、perimental overview:,Limit of Detection: 1 in 30,000 transcripts 20 transcripts/cell,Red increase of Cy5 sample transcripts Green increase of Cy3 sample transcripts Yellow equal abundance,Affymetrix GeneChip,Limits: 1: 100,000 transcripts 5 transcripts/cell,http:/,Affymetrix:,Gene Expression Arrays
8、Transcripts/Genes Arabidopsis Genome 24,000 C. elegans Genome 22,500 Drosophila Genome 18, 500 E. coli Genome 20, 366 Human Genome U133 Plus 47,000 Mouse Genome 39, 000 Yeast Genome 5, 841 (S. cerevisiae) & 5, 031 (S. pombe) Rat Genome 30, 000 Zebrafish 14, 900 Plasmodium/Anopheles 4,300 (P. falcipa
9、rum) & 14,900 (A. gambiae) Barley (25,500), Soybean (37,500 + 23,300 pathogen), Grape (15,700) Canine (21,700), Bovine (23,000) B.subtilis (5,000), S. aureus (3,300 ORFS), Xenopus (14, 400),Microarray and GeneChip Approaches,Advantages: Rapid Method and data analysis well described and supported Rob
10、ust Convenient for directed and focussed studies Disadvantages: Closed system approach Difficult to correlate with absolute transcript number Sensitive to alternative splicing ambiguities,(二)SAGE在转录物水平研究细胞或组织基因表达模式,SAGE的基本原理: 利用锚定酶(anchoring enzyme,AE)和位标酶(tagging enzyme,TE)切割DNA分子的特定位置(一般近3端),分离SAG
11、E标签(长约14 bp,可藉此鉴定基因组中的所有基因),并将这些标签串联起来,然后对其进行测序 特点: 可全面提供生物体基因表达谱信息 可用来定量比较不同状态下组织或细胞的所有差异表达基因,Anchoring Enzyme NlaIII, recognition site: The 3 terminus of adaptor A and B are both TCCRACTAG, where a recognition site of Tagging Enzyme MmeI flanked with NlaIII,Hu M, Polyak K. Nature Protocols 2006,Ta
12、gging Enzyme MmeI recognition site:,Hu M, Polyak K. Nature Protocols 2006,SAGE,Advantages: Potential open system method new transcripts can be identified Accuracy of unambiguous transcript observation Digital output of data Quantitative and qualitative information Disadvantages: Characterising novel
13、 transcripts is often computationally difficult from short tag sequences Tag specificity (recently increased length to 21 bp) Length of tags can vary (TE enzyme activity variable with temperature) A subset of transcripts do not contain enzyme recognition sequence Sensitive to a subset of alternative
14、 splice variants,(三)MPSS是以基因测序为基础的基因表达谱分析新技术,MPSS的原理: 一个含有能够特异识别转录子的信息标签序列(1020 bp)与长的连续分子连接在一起,测出mRNA的一端包含一个10至20个碱基的标签序列。每一标签序列在样品中的频率(拷贝数)代表了与该标签序列相应的基因表达水平。 基因表达水平是以计算mRNA拷贝数为基础,是一个数字表达系统。只要将病理和对照样品分别进行测定,即可进行严格的统计检验,能测定表达水平较低、差异较小的基因,而且不必预先知道基因的序列。,四、RNA组学研究全部snmRNA,人类基因组序列特点:2万2.5万个基因,与蛋白质合成有关
15、的序列占整个基因组的2%左右,其余98%的基因组序列没有得到注释。 RNA组学研究范畴:小分子RNA,包括 snRNA、snoRNA、scRNA、siRNA、miRNA,Small RNA Catalogues Naqvi (2009) Int J Biol Sci,Within cells there are a variety of discovered small RNAs in length in 19-30 nt, recently governing diverse cellular processes such as development, differentiation ac
16、ross the eukaryotic kingdom. siRNA : short interfering RNA as defensive mechanism to protect host genome integrity from intrusion of foreign nucleic acids. miRNA (microRNA) : 21-30 nt in length transcribed from host genome loci, involved in wide cellular processes, specific to development and differ
17、entiation. tasiRNA (trans-acting short interfering RNA) : 21 nt length taken endogenous transcript as template, under RdRP activity, followed by Dicer to produce tasiRNA. In human and fly without tasiRNA it is due to absent to RdRP.,27,Small RNAs Catalogues,rasiRNA (repeat-associated RNA) : 24-26 nt
18、 long products of DCL3 (Dicer like protein, in plant) on long dsRNA formed, usual in retro-transposon loci with methylation, to play role in gametogenesis via recruiting chromatin remodeling proteins to modulate chromatin status. scnRNA (small scanning RNA) : 29 nt long scnRNA, reported from protozo
19、an, derived from micro-nuclei, eliminated original loci of genome, given birth to macro-nuclei. lsiRNA (long siRNA): 30-40 nt in length induced in response to bacterial infection or growth condition. piRNA (piwi-acting RNA) , 21-U RNA,28,MicroRNA 简介, (1)长度为21nt左右核苷酸的内源性单链小分子RNA;(2)存在65nt左右的发夹结构前体;(3
20、)基因座位于蛋白质基因间隔区;(4)其DNA序列在近源物种间高度保守。 miRNA具有十分重要的调控功能,它们主要参与基因转录后水平的调控 。能够通过与靶mRNA特异性的碱基配对引起靶mRNA的降解(植物中较为常见)或者抑制其翻译(动物中较为常见),从而影响了靶mRNA的表达。 目前发现miRNA是一个庞大的小分子调控RNA家族,广泛存在于各种动植物中,参与细胞增殖和分化、细胞凋亡、胚胎发育、形态建成以及疾病发生等一系列重要的生命过程。 最近发现一系列与肿瘤发生相关的和人类病毒编码的miRNA,揭示miRNA在哺乳动物基因表达调控中具有重要作用。,30,Rana (2007) J Cell P
21、hysiol,*Small RNA biogenesis - RISC formation - RNAi & Its Mechanism - Cell Phenotypy*,31,32,Rana (2007) Nature Rev Mol Cell Biol,33,*Overview on RNA Interference,Genomic loci transcribed by RNA Pol II, III and IV to form double-stranded RNAs (dsRNA) or viral RNA dependent RNA polymerase (RdRP) to g
22、enerate dsRNA. DsRNA trimmed by RNase III (Drosha in nuclear and Dicer in cytoplasm) to small duplex RNA with 19-30 bp. A single stranded RNA unwound by Argonaute as guide RNA and loaded into RISC (RNA induced silencing complex). Complementary to target RNA by guide RNA in RISC and triggered RNA int
23、erference.,RNA Interference Target RNA to be degradation. Target RNA to be translational repression or destabilization. Target RNA to be transcriptional repression. The genomic locus of target RNA to become heterochromatin or degradation.,34,*Biogenesis of miRNAs and siRNAs Bartel (2004) Cell,36,*Bi
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- 转录 transcriptomicswxj
链接地址:https://www.31doc.com/p-3011911.html