微珠芯片.ppt

DNA RNA Protein 基因表达是根据基因的DNA模板进行mRNA和蛋白质合成的过程，各种基因的表达存在差异，一种组织中基因表达水平的差异可达1万倍。功能基因研究的一种重要的方法就是采用高通量基因表达检测技术，全面分析基因的表达水平，了解基因的功能。 人类全基因组芯片I Human-6 6 样品/芯片 48,000 序列 10 million 特异探针 实验方法—单轮IVT反应 RNA 分离 RT cDNA 合成 IVT biotinylated cRNA SA-Cy3杂交 完整RNA检测:直接杂交 性能:灵敏度-动力学范围-精确性 性能:准确性(与qPCR 比较) DASL 检测 分析 福尔马林固定或 Paraffin-Embedded Tissue 石蜡包埋样品（FFPE) DASL 检测  DASL vs qPCR DASL vs qPCR 完整 RNA vs 降解 RNA 低浓度 vs 高浓度 文献：临床样品研究 文献:肿瘤标记基因的发现 DASL? ：商品化肿瘤 芯片 DASL 总结 高通量检测FFPE样品中的基因表达 样品起始浓度: 200ng 总 RNA 同时检测约500 genes (每个基因3-4 探针) 同时可处理 16 或96个样品 高灵敏度，可检测出极小的样品差异 基因表达谱研究 SIM2 Expression 0 200 400 600 800 1000 1200 1400 1600 1800 2000 fresh-frozen 1 fresh-frozen 2 FFPE 1 FFPE 2 FFPE 3 FFPE 4 FFPE 5 FFPE 6 FFPE 7 FFPE 8 Samples Intensity cancer normal 微珠芯片应用---基因表达谱研究 DeYoung et al., 2003. PNAS 100: 4760-5 五百多个肿瘤相关基因 Human Sampler 特定基因芯片 用户定制芯片 FFPE 组织切片 基因表达 芯片 (DASL!) 全基因组表达芯片 肿瘤 定制 10,000’s 100’s Genes Human-6 Human Ref-8 微珠芯片应用---基因表达谱研究 降解RNA研究芯片 在定义上把我们和 传统的点样芯片和光原位合成芯片分开 Fully custom and bioinformatically-optimal 50mers are designed, synthesized, and immobilized to beads. The oligo used is actually a 73mer, which includes a concatenated 23-mer illumiCode oligo for decoding. Noteworthy: the actual oligo synthesis begins with probe and ends with the illumiCode, with the amine (used for attachment to bead) added at the end of the illumiCode. This ensures that any bead that is successfully decoded also contains a gene-specific probe. This does not protect against deletions, however deletions in Oligator oligo synthesis are exceedingly rare, and oligos are routinely checked by MS We use 2 probes per gene to maximize performance. 98% of your genes will have 2 probes in each array 100% of genes will have at least one probe in each array. The assay uses standard IVT sample labeling, with reagents from Ambion and Qiagen that researchers are accustomed to using. Only a single round of amplification is required A reverse tra