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1、Microarray Analysis 郑 芳 Fang Zheng Ph.D. Prof. Tianjin Medical University 1 Presentation outline Introduction DNA Microarray Tissue Aarray Analysis Flowarray Analysis 2 Presentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis 3 Definition of Microarray Microarray Ana
2、lysis: Introduction 生物芯片： 能对生物成分或生物分子进行快速并行处理和分 析的技术。 检测原理：利用特异性的分子间的相互作用， 如核酸杂交、抗原-抗体特异性结合、蛋白-蛋白 间特异性结合等。 4 1. General Introduction If you get the gene, you will get the protein. If you want a protein, get its gene! 5 Abnormality of Gene structure Abnormality of Gene expression Central dogma Techno
3、logy PCR RE analysis Hybridization DNA sequencing DNA fingerprinting DNA microarray RT-PCR FQ-PCR Northern blot DNA microarray (Gene chip) Western-blot IHC stain ELIZA Protein chip Tissue Array DNAmRNAProtein transcriptiontransplantation RT 6 History of Microarray development Southern HGP：To identif
4、y and determine DNA sequence of human genome Sequencing by hybridization, SBH (杂交测序)： 建立在特定序列的DNA与特定长度的寡核苷酸集合的杂 交的基础之上。未知DNA序列可以通过鉴定与靶DNA序列形 成完整的双链体寡核苷酸的重叠区域来确定. 65536个八核苷酸点阵可测定约200个核苷酸的序列； 一百万个十二核苷酸点阵可测定约1000个碱基对. Microarray Analysis Applications of biochip technologies 16 3. Search new genes 4. Mu
5、tation and polymorphism analysis l To detect and analyze DNA variation and polymorphism on a large scale l Detection of genetic mutation l Inherited diseases, and tumor Microarray Analysis Applications of biochip technology 17 5.Drug screening 在基因水平上寻找药物靶标，毒性对基因的影响。 临床许多细菌对药物具有抗药性，但是不同的亚型对 同一药物的敏感性不
6、同。DNA芯片技术已被用于鉴定结 核菌及非典型分支杆菌的基因型与耐利福平的相关性以 及HIV产生抗药性与其逆转录酶及蛋白酶基因突变的相 关性。这为临床选择敏感药物提供了有效手段。 Applications of biochip technology Microarray Analysis 18 6. Proteinogram analysis 基因组计划后的另一项重要计划-蛋白组(Proteomics) 计划. Contrast analysis of proteinogram on normal and cancer cells by Protein chips Applications o
7、f biochip technology Microarray Analysis 7. Miniaturization of analytical apparatus 19 Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Schena, M., et al., Science 1995;270:467470 Parallel human genome analysis: Microarray-based expression monitoring of 1000 g
8、enes. Schena, M., et al., PNAS 1993:1061410619 Use of a cDNA microarray to analyse gene expression patterns in human cancer. DeRisi, J., et al., Nature Genetics 1996;14:457460 References: Microarray Analysis Applications of biochip technology 20 21 22 23 Presentation outline Introduction DNA Microar
9、ray Tissue Array Analysis Flowarray Analysis 24 DNA Microarray Analysis Principle of Microarrays DNA芯片技术是指在固相支持物上原位合成（in situ synthesis）寡核苷酸探针，或者直接将大量的DNA探针以 显微打印的方式有序的固化于支持物表面，然后与标记的 样品杂交，通过对杂交信号的检测分析即可得出样品的遗 传信息（基因序列及表达的信息）。 又被称为基因芯片（gene chips）、DNA阵列（DNA array）、cDNA芯片（cDNA chips）、寡核苷酸阵列（ oligonuc
10、leotide array）等。 25 DNA Microarray Analysis Principle of Microarrays Microarray assays are based on hybridization of a single-stranded DNA labeled with a fluorescent tag to a complementary molecule attached to the chip 26 lMicroarrays enable simultaneous measurement of the expression levels of thous
11、ands of genes in a sample lMicroarray: nGlass slide with a matrix of thousands of spots printed on to it nEach spot contains probes which bind to a specific gene DNA Microarray Analysis Principle of Microarrays 27 lConstruction Place array of probes on microchip Probe is oligonucleotide 25 bases lon
12、g that characterizes gene or genome Chip is about 2cm by 2cm Solid support (glass, plastic, metal, silicon) Hybridized probes (DNA molecules) are fluorescently labeled DNA Microarray Analysis Principle of Microarrays 28 lWhen each spot in a microarray is attached a unique DNA molecule, it can be use
13、d to detect presence/absence or even concentration of a particular type of DNA molecule in test tube. DNA Microarray Analysis Principle of Microarrays 29 p Chip preparation 芯片制备 p Sample preparation 样品制备 p Work on the biochemical principle of DNA/DNA hybridization 杂交反应 pAnalyze hybridization pattern
14、 结果分析 DNA Microarray Analysis Basic idea Basic idea of Microarrays 30 DNA Microarray Analysis Basic idea Basic idea of Microarrays 31 lGene expression lGene disease relation lGene-gene interaction lFinding Co-Regulated Genes lUnderstanding Gene Regulatory Networks l DNA Microarray Analysis One gene
15、at a time before, now tens of thousands simultaneously 32 DNA Microarray Analysis Microarrays designs and applications: cDNA microarrays in gene expression studies cDNA Microarray cDNA clone inserts are printed onto glass slides at high density As many as 25,000 cDNAs can be applied to a single glas
16、s slide Enables large-scale, high- throughput analysis 33 DNA Microarray Analysis cDNA Microarrays Thousands of known genes are available Requires special instrumentation for generation of microarrays and for analysis of results Microarrays designs and applications: cDNA microarrays in gene expressi
17、on studies 34 DNA Microarray Analysis Sample preparation and hybridization to cDNA microarrays 35 DNA Microarray Analysis Comparison of Gene Expression Patterns Between Normal Lung and Lung Carcinoma for Identification of Genes Associated with Neoplastic Transformation NormalTumorNormalTumor Patient
18、 1Patient 2 36 l用于基因转录水平的检测、基因组分 析和后基因组研究 美国斯坦福大学Davis等用PCR法从外周血淋巴细胞cDNA 文库中得到了1046种cDNA片段，制成芯片。然后与热处 理的T淋巴细胞和未处理的T细胞种提取的mRNA反转录出 的单链cDNA片段杂交。经激光共聚焦扫描系统分析，共 发现17个差异表达的基因，其中11个是被热诱导的，6 个是被热抑制的。经序列分析证实，其中3个是未报导 的新基因。 DNA Microarray Analysis DNA microarrays application 37 Microarray Analysis: Developme
19、nt Microarray designs and applications Microarray Probes cDNAs (from libraries or PCR products) Oligonucleotides Genomic DNA fragments 38 Microarray Analysis: Development Microarray designs and applications Applications Gene expression studies Oligo-mediated sequencing Genetic mapping studies Mutati
20、on analysis Polymorphism analysis 39 The tools provided to us by our understanding of molecular biology, are what allow us to interrogate DNA/RNA for the purpose of identifying disease causing by genetic alterations. DNA Microarray Analysis DNA microarrays application 40 Presentation outline Introdu
21、ction DNA Microarray Tissue Array Analysis Flowarray Analysis 41 Tissue Array Analysis Tissue microarrays 1998 Kononen, Nature Medicine allow one to identify and evaluate highly specialized gene expression patterns and thus are a high-throughput tool (for diagnosis). Tissue Microarray DNA Microarray
22、 Tissue microarraysTissue microarrays 42 Complementary uses of DNA and tissue arrays l DNA microarrays for gene expression Used to find biomarkers Relatively few tumor samples but thousands of markers (genes) l TMAs for protein expression patterns Used to validate biomarkers large number (hundreds)
23、of tumors, relatively few markers Tissue Array Analysis 43 TMAs often used for validating tumor genes in cancer research l The current classification systems of tumors are partially predictive of outcomes, and are based primarily upon morphologic criteria (grade, stage) l The hope is that tumor mark
24、ers may lead to improved diagnostic, prognostic and therapeutic applications in the clinic. Tissue Array Analysis 44 donor block array block slide Tissue Microarray (TMA) Technology Kononen et al. Nature Medicine 1998 Tissue Array Analysis 45 Tissue Microarray (TMA) Technology Hundreds of tiny (typi
25、cally 0.6 mm diameter) cylindrical tissue cores -densely and precisely arrayed into a single histologic paraffin block. From this new array block, up to 300 serial 4-8 m thick sections may be produced. Targets for protein expression by immuno- histochemical studies and fluorescence in situ hybridiza
26、tion (FISH). Tissue Array Analysis 46 Tissue Array Section 0.6 mm 0.2mm 700 Tissue Samples Tissue Array Analysis 47 Real picture: brown staining by D. Seligson Tissue Array Analysis 48 Ki-67 Expression in Kidney Cancer High GradeLow Grade Message: brown staining related to tumor grade Tissue Array A
27、nalysis 49 Multiple measurements per patient: Several spots per tumor sample and several “scores” per spot Maximum intensity = Max Percent of cells staining = Pos Percent of cells staining with the maximum intensity = PosMax Spots have a spot grade: NL,1,2, Indicator of missingness Each patient (tum
28、or sample) is usually represented by multiple spots 3 tumor spots 1 matched normal spot Tissue Array Analysis 50 Properties of TMA Data lHighly skewed, non-normal, semi- continuous. nOften a good idea to model as ordinal variables with many levels. lStaining scores of the same markers are highly cor
29、related Tissue Array Analysis 51 Presentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis 52 Flowarray Analysis Basic knowledge of flow cytometry 53 + 流式分析 Flow cytometric analysis 捕获标记 待测配体检测探针 包被探针 Coating probe 荧光 微载体 microcarrier Flowarray Analysis Working princi
30、ple of flowarray analysis 54 l Principle： 用高聚分子微粒为载体, 在微载体表面 进行探针-配体杂交, 用流式细胞仪检测标 记抗体的荧光强度. Flowarray Analysis Working principle of flowarray analysis 55 l Technique characteristic： 微量化、数字化、图像化和高通量化 microamount, digitalization, topography and high throughput l Application： Research, clinical diagnos
31、is, drug development Flowarray Analysis Working principle of flowarray analysis 56 Principle of high throughput analysis Flowarray Analysis (From Prof. Y LIN) 57 n BD CBATM液态芯片（科研）试剂盒（美国 2001） n Luminex 100液态芯片（科研）试剂盒（美国2004 ） n FlowChipTM流式芯片试剂盒（西安微通2007） 临床诊断 免疫监测 药物研发 current situation of Flowarr
32、ay technology Flowarray Analysis (From Prof. Y LIN) 58 RF-IgARF-IgM Normal RA Semi-works production 1: Rheumatoid factor Flowarray Analysis 59 Y052405.007 100101102103104 FL2-H Y052405.008 100101102103104 FL2-H Y052405.020 100101102103104 FL2-H Y051705IL2.004 100101102103104 FL2-H Y051705IL2.005 100
33、101102103104 FL2-H Y051705IL6.004 100101102103104 FL2-H Y051705IL6.005 100101102103104 FL2-H Y052405.019 100101102103104 FL2-H 肾移植无排斥肾移植排斥 IL-5 IFN- IL-6 IL-2 Semi-works production 2: organ-graft refection Flowarray Analysis (From Prof. Y LIN) 60 61 Thank you Thank you for your for your attention attention ! ! 62