miRNA研究整套方案

microRNA研究的整体解决 方案 气瓶 现场处置方案 .pdf气瓶 现场处置方案 .doc见习基地管理方案.doc关于群访事件的化解方案建筑工地扬尘治理专项方案下载 & 最新进展：TaqMan assays Amy Guo过健英 amy.guo@appliedbiosystems.com 2 © 2008 Applied Biosystems 小RNA研究—— Hot topics in the world ● 2000年，RNAi的研究进展被Science杂志评为重大科技突破。 ● 2001年，RNAi作为当年最重要的科学研究成果之一，再次入选“十大科技突破”。 ● 2002年12月20日，Science杂志将“Small RNA & RNAi”评为2002年度最耀眼的明 星。同时，Nature杂志亦将Small RNA评为年度重大科技成果之一。 ● 2003年，microRNA的研究第四次入选“十大科技突破”，排在第四位。 ● 2005年，microRNA的研究第五次入选“十大科技突破”。 ● RNA研究的突破性进展，是生物医学研究领域近20年来，可与HGP相提并论的最 重大成果之一。 3 © 2008 Applied Biosystems microRNA基因组分布 ~62% are Intergenic ~34% are Intronic ~4% are Exonic ~15% of miRNAs in clusters miRNAMap: http://mirnamap.mbc.nctu.edu.tw/ Hsu, Huang, Hsu, Lin, Tsou, Tseng, Stadler, Washietl and Hofacker (2006), NAR 34:D135-139 4 © 2008 Applied Biosystems miRNA 的产生与功能 1. 在细胞核内转录成前体转录本 miRNA) 2. 被RnaseⅢ核酸酶 Drosha加工 成约70-90nt的发夹状 pre-miRNA 3. 转运到细胞质被Dicer加工成成熟 的 miRNA 4. 双链 miRNA分子被解链，单链的 miRNA进入一个核糖蛋白复合体 miRNP ( RISC) 5. 通过与靶基因的3' UTR 区互补配 对，指导 miRNP复合体对靶基因 mRNA 进行切割或者 翻译 阿房宫赋翻译下载德汉翻译pdf阿房宫赋翻译下载阿房宫赋翻译下载翻译理论.doc 抑制。 *mature active strand 5 © 2008 Applied Biosystems microRNA ● 成熟的MicroRNA(miRNA)是一种 22个nt左右的内源性单链小分子 RNA ● 由带茎环结构的双链RNA前体剪切 而成 ● 具有高度保守性、时序性和组织特 异性 ● 通过与特异mRNA结合，抑制目标 基因表达或降解mRNA ● 调节人类三分之一的基因 6 © 2008 Applied Biosystems miRNA在动物与植物中的功能 • 发育 • Timing (Lee et al. 1993) • Cell proliferation (Brennecke et al. 2003) • Stem cell & neuron (Krichevsky et al. 2003; Hatfield et al., 2005) • 细胞死亡 (Baehrecke, 2003) • 疾病 • 肿瘤 (Lu et al. 2005) • 原癌miRNA (He et al. 2005) • 脑肿瘤 (Ciafre et al. 2005; Chan et al. 2005) • 白血病 (Calin et al. 2002; 2004) • 糖尿病 (Poy et al. 2004) • 胆固醇的生物合成 (Krutzfeldt et al. 2005) • DNA 甲基化与染色质修饰 (Bao et al. 2004) 7 © 2008 Applied Biosystems 哪些重要的miRNA参与 调控了某个生物过程? 表达了什么miRNA? 分离, 克隆, 测序 miRNA文库筛选 何时何处表达miRNA? miRNA表达分析 miRNAs控制了 那些基因 ? 功能研究 治疗 miRNA研究的基本问 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 诊断 8 © 2008 Applied Biosystems miRNAs与疾病关系的研究 miRNA表达的分析 Compare miRNA profiles of cancer and normal samples miRNA功能研究 Identify miRNAs that affect cell division, apoptosis, angiogenesis, cell proliferation, telomerase activity, etc. 0 25 50 75 100 125 150 175 200 在疾病样本中差异性表达的 miRNAs 诱导或消除了疾病表型的 miRNAs 靶基因 9 © 2008 Applied Biosystems 分离、纯 化/富集 microRNA 1 STEP Examine biological impact of silencing the target Correlate silencing with biology STEP STEPSTEP microRNA功 能研究：调 节靶基因的 鉴定 3 表达分析： microRNA 的检测与定 量 STEP 2 10 © 2008 Applied Biosystems mirVana™ miRNA Isolation Kit ● 适合各种组织和细胞材料 ● 专业高效回收小分子RNA ● 富集小于200 nt RNA，提高下游试验灵敏度 ● 方便快捷，30分钟完成整个过程 ● 理想的 miRNA, siRNA, shRNA, 和snRNA分析试剂盒 11 © 2008 Applied Biosystems 不同样本中miRNA的纯化方案 12 © 2008 Applied Biosystems 分离、纯 化/富集 microRNA 1 STEP Examine biological impact of silencing the target Correlate silencing with biology STEP STEPSTEP microRNA功 能研究：调 节靶基因的 鉴定 3 表达分析： microRNA 的检测与定 量 STEP 2 13 © 2008 Applied Biosystems miRNA检测的技术挑战 Precursor miRNA Northern Blot (杂交法) • 常规 方法 快递客服问题件处理详细方法山木方法pdf计算方法pdf华与华方法下载八字理论方法下载 ，易于操作 • 需大量样品(ug级) • 动力学范围低（约2个数量级） • 错配杂交问题，难以区分通常只有很少序列差别的序列 Microarrays (芯片法) • 通量高 • 可测动力学范围较宽 • 需大量RNA样品，约5 µg per array • 很难区分前体miRNA and 成熟miRNA • 对类似结构的miRNA分辨率差 Quantitative Real-Time PCR (实时定量PCR) • 只需微量样品 • 非常宽的动力学范围（7 log） • 高灵敏度, 高特异性 • 但是, 如何设计这样一个靶物只有22个碱基TaqMan real-time PCR? Solution: TaqMan® MicroRNA PCR Assays Mature miRNA 14 © 2008 Applied Biosystems miRNA RT primer Step 1: Stem-loop RT Step 2: Real-time PCR cDNA Forward primer Reverse primer FQ TaqMan probe TaqMan® MicroRNA Assays 成分： 1. RT primer 2. Forward primer 3. Reverse primer 4. TaqMan probe 优点 ‹ 高特异性，能区分前体和成 熟miRNA。 ‹ 高灵敏度， 1-10ng样本。 ‹ 精确度高，动态范围宽，不 同拷贝都能检测。 ‹ 快速-- 3小时。 Nucleic Acid Res. v33: e179, 2005 15 © 2008 Applied Biosystems Stem-loop RT-PCR miRNA assays 高度的重现性 let-7a miR-20 miR-324-5 miR-16 4个miRNAs靶点， 16 个重复， 2名操作者的实验中 平均 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 方差: 0.1 定量结果偏差: 0.6% 16 © 2008 Applied Biosystems 宽动态范围——可以达到7个数量级的动态型范围 17 © 2008 Applied Biosystems 以TaqMan®探针为基础的microRNA检测 ● 按检测通量分： – 单个/少量microRNA的检测 – 中到高通量microRNA的检测 – 少量细胞/大量样本 ● 整个实验所需要的试剂包括： – 反转录引物、定量PCR引物与探针 • TaqMan microRNA assay • TaqMan microRNA Array + Megaplex™ RT Primers/Primer Pools – 反转录试剂：TaqMan MicroRNA RT Kit 200 rxns (20ul) 4366596 – 定量PCR试剂：TaqMan Universal Master Mix, No UNG (5ml) 4324018 – TaqMan microRNA Cell-to-Ct 18 © 2008 Applied Biosystems I. 单个/少量目标microRNA的检测 •TaqMan microRNA assay •反转录试剂 TaqMan MicroRNA RT Kit 200 rxns (20ul) 4366596 •定量PCR试剂 TaqMan Universal Master Mix, No UNG (5ml) 4324018 19 © 2008 Applied Biosystems 单个的TaqMan® MicroRNA Assays Version 10 ● Individual Assays Now Available – 692 Human • Plus 18 Endogenous controls – 544 Mouse • Plus 10 Endogenous controls – 342 Rat – 82 C. elegans – 65 Arabidopsis – 72 Drosophila – Additional assays released regularly 20 © 2008 Applied Biosystems 在线搜索miRNA assays? ——www.appliedbiosystems.com 关键词搜索 批量搜索 种属选择 建议按照target sequence搜 索，而不是microRNA编号 21 © 2008 Applied Biosystems II. 中到高通量microRNA的检测 New • TaqMan microRNA Array + Megaplex™ RT Primers/Primer Pools •反转录试剂 TaqMan MicroRNA RT Kit 200 rxns (20ul) 4366596 •定量PCR试剂 TaqMan Universal Master Mix, No UNG (5ml) 4324018 22 © 2008 Applied Biosystems Case Study ● 应用TaqMan microRNA表达谱分析技术 研究白血病相关的microRNA生物标记物 Stanford Institute for Regenerative Medicine and Stem Cell Biology 23 © 2008 Applied Biosystems Human Leukemia Stem Cells (LSC) Tan BT, Park CY, Ailles LE, Weissman IL. Lab Invest. 2006. 24 © 2008 Applied Biosystems Big Questions in Leukemia Stem Cell Biology Similarity Leukemia LSC Normal HSC Difference ● Can we identify molecular markers to better define and characterize the HSC and LSC? – Surface markers – mRNA ? – microRNA ? ● What are the molecular mechanisms underlying the normal HSC and leukemia stem cell? – Genetic – Transcription – Epigenetic control – Posttranscriptional regulation (i.e. microRNA) 25 © 2008 Applied Biosystems Putative miRNA Roles in Hematopoietic Cells Expression Target Normal hematopoiesis 1) miR-142 ⇑ B and myeloid lineage 2) miR-155 ⇑ GC B cells and activated T cells 3) miR-181a ⇑ B lineage 4) miR-221 ↓ Erythropoiesis KIT receptor 5) miR-222 ↓ Erythropoiesis KIT receptor 6) miR-223 ⇑ Myeloid lineage, granulopoiesis NFI-A 7) miR-155 ↓ Erythropoiesis, ↓ myelopoiesis, 8) miR-150 ⇑ B lineage Hematopoietic malignancies 1) miR-15a ↓ CLL BCL-2 2) miR-16-1 ↓CLL BCL-2 3) miR-17-92b ⇑ DLBCL E2F1 4) miR-125b-1 Involved in translocation in B-ALL case 5) miR-142 Involved in translocation in B-PLL case 6) miR-155 ⇑ HL, DLBCL, PMBL, PTLD and CLL, ↓ BL Modified from Kluiver, et al. Leukemia 2006;20:1931-1936. 26 © 2008 Applied Biosystems MicroRNA Profiling in HSC/Progenitors and LSC ● FACS-purified cell populations – Normal HSC and committed progenitor cells from bone marrow of 13 normal donors (<35 y/o, HIV/HSV serology negative, otherwise good health). • HSC, CMP, CLP,GMP and MEP – AML LSC and blasts from bone marrow of 12 independent, newly diagnosed AML patients • CD34+CD38-Lin- leukemia stem cell (LSC) • CD34+CD38+ leukemia blasts (non-LSC) 27 © 2008 Applied Biosystems 0 102 103 104 105 0 102 103 104 105 99.4 0 102 103 104 105 0 102 103 104 105 69.4 4.63 0 102 103 104 105 0 102 103 104 105 63 ● CD14 ● CD20 ● CD19 ● CD3 ● CD4 ● CD8 ● CD56 ● GPA Lin CD90 CD34 C D 3 8 S S C P I 0 102 103 104 105 0 102 103 104 105 0 99.5 CD34 C D 3 8 CD34 C D 3 8 0 102 103 104 105 0 102 103 104 105 99.8 0 Lineage Antibodies LSC Non-LSC AML LSC can be highly-purified by FACS (0.1-1%) 28 © 2008 Applied Biosystems MegaPlex RT Primers/Primer Pools Total RNA cDNAs MegaPlex RT (up to 300+plex) Amplified cDNAs MegaPlex Pre-Amplification 12 cycles PCR ● 从少量，甚至是单细胞中进行 microRNA表达谱分析 FQ TaqMan® Real-time PCR 29 © 2008 Applied Biosystems Correlation Between With or Without Preamplification ddCt_brain_kindey_preAmp -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 30 © 2008 Applied Biosystems TaqMan® MicroRNA Arrays •快速，高效的miRNA检测方法 •同时检测多达365个miRNA 31 © 2008 Applied Biosystems Distinct miRNA Expression Patterns Characterizing AML, HSC and Progenitors ● 62 samples, 318 miRNA analyzed ● 83 genes differentially expressed – SAM, FDR < 1% – Ct < 30 in > 25% samples ● Unsupervised hierarchical clustering -3 0 3 AML Progenitors HSC CMP CLP GMP MEP HSC LSC Non-LSC HSC/Prog HSC AML HSC/AML Prog miR-17-5pmiR-20a miR-106a 32 © 2008 Applied Biosystems miRNA Expression Patterns Characterizing Similarity and Difference Between HSC and LSC ● 54 samples, 318 miRNA analyzed ● 95 genes differentially expressed – SAM, FDR < 1% – Ct < 30 in > 25% samples ● Supervised hierarchical clustering LSC ProgenitorsHSC CMP CLP GMP MEP HSC LSC HSC HSC/ LSC (Stemness) Prog LSC HSC/Prog (normal) miR-181a miR-155 -3 0 3 33 © 2008 Applied Biosystems Version 11TaqMan microRNA arrays Human A+B Rodent A+B 34 © 2008 Applied Biosystems III. 少量细胞/大量样本的microRNA检测 •TaqMan microRNA Cell-to-Ct •TaqMan microRNA Assay ---少量microRNA靶点 或 •TaqMan microRNA Array + Megaplex™ RT Primers/Primer Pools --- microRNA表达谱分析 35 © 2008 Applied Biosystems TaqMan® MicroRNA Cells-to-CT™ Kit： 4391848 100 Lysis reactions with gDNA removal 200 miRNA cDNA synthesis reactions (200 μL) 500 TaqMan UMM PCR (5 mL) 4391996 400 lysis reactions with gDNA removal 1,000 miRNA RT (1,000 μL) 2,000 TaqMan UMM PCR (20 mL) z 直接从细胞开始定量检测microRNA的完全解决方案 z 适合10 - 100,000个培养细胞 z 简单、快速，无需传统的RNA分离/抽提 z 以TaqMan探针技术为基础，microRNA分析的黄金标准 36 © 2008 Applied Biosystems 1.裂解 • 全部室温下进行 • 无需离心 • 无有机溶剂 • 不需过滤，无阻塞之虞 • 可以在96 /384孔板上直接进行 2.反转录 • 多至33%的裂解产物可用于反 转录 • 可以与Multiplex配合使用 3.定量PCR • 配足5 PCR/每次裂解 TaqMan® MicroRNA Cells-to-CT™ Kit 快速，简单 37 © 2008 Applied Biosystems TaqMan® MicroRNA Cells-to-CT™ Kit: 以TaqMan探针技术为基础，microRNA分析的黄金标准 与纯化的RNA相同的检测效果 38 © 2008 Applied Biosystems 宽动态范围 –线性检测跨10到 100,000 细胞 Q Q • 结果显示这两个目标microRNA检测的线性与灵敏度跨越5个对数级 – 与 纯化RNA的结果相同 39 © 2008 Applied Biosystems 分离、纯 化/富集 microRNA 1 STEP Examine biological impact of silencing the target Correlate silencing with biology STEP STEPSTEP microRNA功 能研究：调 节靶基因的 鉴定 3 表达分析： microRNA 的检测与定 量 STEP 2 40 © 2008 Applied Biosystems miRNA功能研究 2，抑制表达的方法：反义寡 核苷酸 (Meister 2004 and Hutvagner 2004) 1，过量表达的方法：转染前 体miRNAs 方法: 用特定的试剂改变内源 miRNAs的水平 PrePre--miRmiR™™ AntiAnti--miRmiR™™ 41 © 2008 Applied Biosystems How do miRNAs affect proliferation? Block miRNA andBlock miRNA and induce target induce target mRNA translation Measure cell Measure cell number number mRNA translation Introduce miRNA inhibitors Anti-miR™ Reducing let-7 Activity Increases Proliferation in A549 Cells 42 © 2008 Applied Biosystems 0 50 100 150 200 250 Let-7a Let-7b Let-7c Let-7d Let-7g m ir-1 m ir-7 m ir-9 m ir-10a m ir-10b N C 1 m ir-15a m ir-16 m ir-17-3p m ir-18 m ir-19a m ir-20 m ir-21 m ir-22 m ir-23a m ir-23b N C 1 m ir-24 m ir-25 m ir-26a m ir-27a m ir-28 m ir-29a m ir-30a-3p m ir-31 m ir-32 m ir-34a N C 2 m ir-95 m ir-96 m ir-92 m ir-98 m ir-99a m ir-100 m ir-101 m ir-103 m ir-105 m ir-107 N C 2 m ir-108 m ir-122 m ir-124 m ir-125a m ir-125b m ir-126 m ir-128 m ir-129 m ir-132 m ir-133A G A PD H m ir-133B m ir-134 m ir-135 m ir-136 m ir-137 m ir-139 m ir-140 m ir-141 m ir-142 m ir-143 G A PD H m ir-144 m ir-145 m ir-146 m ir-147 m ir-148 m ir-149 m ir-150 m ir-151 m ir-152 m ir-153 E m pty m ir-155 m ir-181a m ir-182 m ir-183 m ir-184 m ir-186 m ir-187 m ir-188 m ir-190 m ir-191 E m pty R e l a t i v e P r o l i f e r a t i o n Pre-miR™ Proliferation Screen Measure cell Measure cell number Increase overall miRNA levels and Increase overall miRNA levels and block target mRNA translation number block target mRNA translation Introduce mature miRNA Pre-miR™ 43 © 2008 Applied Biosystems Identifying miRNA targets 1.1. 2.2. 3.3. 4.4. miRNA features:miRNA features: 1. Seed region, perfect base match from position 2-8 2. Center bulge, no more than 8 bases 3. G-U pairing on the 3’ end, extend for 22 bases and match at least 6 4. Overall duplex stability (∆G) between miRNA and mRNA Filters Filters • Target-binding site conservation across multiple species • Restrict search to 3’ UTRs • Focus on relevant genes – i.e. known oncogenes 44 © 2008 Applied Biosystems Predicting onco-miR targets Johnson Johnson et alet al, , Cell,Cell, March 2005March 2005 miRNAmiRNA Predicted Gene TargetPredicted Gene Target miR-21 mutS homolog2 (MSH2) miR-21 v-ski sarcoma viral oncogene (SKI) miR-143 breakpoint cluster region (BCR) miR-143 MCF.2 derived transforming sequence (MCF2) miR-143 von Hippel-Lindau tumor suppressor (VHL) miR-143 v-Ki-ras2 sarcoma oncogene (KRAS2) miR-143 Cas-Br-M retroviral transforming sequence (CBL) miR-145 Cas-Br-M retroviral transforming sequence (CBL) miR-188 v-myc viral oncogene (MYCL1) miR-200b cadherin 13 (CDH13) miR-219 v-myc myelocytomatosis viral oncogene (MYCL1) miR-219 B-cell CLL/lymphoma (BCL2) miR-219 cadherin1 (CDH1) let-7 nRAS, kRAS, nRAS (multiple binding sites) let-7 MYC (multiple binding sites) 45 © 2008 Applied Biosystems Block miRNA andBlock miRNA and induce target induce target mRNA translationmRNA translation negative controlnegative control letlet--7 Anti7 Anti--miRmiR™™ InhibitorInhibitor HeLaHeLa cells (high let7)cells (high let7) Anti-miR™ Confirmation of let-7 target genes miRNA Subtraction Experiment MYCMYC protein RASRAS protein Introduce miRNA inhibitors Measure increased Measure increased protein levelsprotein levels 46 © 2008 Applied Biosystems negative controlnegative control letlet--7 Pre7 Pre--miRmiR™™ PrecursorPrecursor HepG2 cells (low let7)HepG2 cells (low let7) Pre-miR™ Confirmation of let-7 target genes miRNA Addition Experiment MYCMYC protein RASRAS protein Increase overall Increase overall miRNA levels and miRNA levels and block target block target mRNA translationmRNA translation Introduce mature miRNA Measure remaining Measure remaining protein levelsprotein levels 47 © 2008 Applied Biosystems Let-7 acts via regulation of RAS 3′ UTR (Johnson et al, Cell March 2005) B.B. Repression of Repression of luciferaseluciferase/RAS 3/RAS 3’’ UTRsUTRs by letby let--77 HeLa (high endogenous let-7) C.C. Induction of Induction of luciferaseluciferase/RAS 3/RAS 3’’ UTRsUTRs by letby let--7 inhibitor7 inhibitor HeLa (high endogenous let-7)A.A. RAS 3RAS 3’’ UTRsUTRs cloned into cloned into pMIRpMIR--REPORTREPORT™™ 48 © 2008 Applied Biosystems microRNA研究的完整解决方案 MegaPlex RT & TLDA MicroRNA Cell-to-Ct 49 © 2008 Applied Biosystems Ambion miRNA Resources www.ambion.com/miRNA 中文指南，请登录生物 通/AB中文网站 新版纸制指南即将推出