专题一基因芯片技术.pptVIP

GeneChipExpression Assay Applications in Genomics Research GeneChip Expression AssayOutline Background  胰岛素表达的机制 GeneChip? Probe Arrays Important Characteristics of Microarray Technology 精确 – Reproducible, specific, quantitative 灵敏 – Few cells, rare RNAs 涵盖面广 – Large numbers of genes in parallel 高通量 – Millions of datapoints/experiment 易于应用 – Exportable, routine use in the lab The GeneChip? Expression Assay Overview of Target Preparation First Strand Synthesis Second Strand Synthesis Second Strand Synthesis Second Strand Synthesis Second Strand Synthesis Second Strand Synthesis In vitro Transcription (IVT) In vitro Transcription (IVT) Fragmentation Fragmentation: End Result 片段化图片 Hybridization Components: Fragmented cRNA Hybridization controls(BioB,C,D, CreX) Blocking agents Salt buffers GeneChip? Probe ArraysHybridization and Staining Files Generated from Expression Assay Expression Analysis File Types Files generated from expression assay .exp file Files generated from expression assay .dat file Files generated from expression assay .cel file Files generated from expression assay .chp file Files generated from expression assay .rpt file Some aspects of the Report file will determine whether our experiment was successful Background: Avg: 43.22 Std: 1.15 Min: 39.40 Max: 46.00 Noise: Avg: 2.39 Std: 0.13 Min: 1.90 Max: 2.80 Background and noise should be low. Otherwise they may overshadow signal from cRNA. Report file(cont) Housekeeping ControlsIf sample preparation was successful housekeeping controls should be present with 3’/5’ of 3.0 What does 3’/5’ mean? What does 3’/5’ mean? High 3’/5’ indicates incomplete transcript Report file(cont) Percentage PresentIf sample preparation was successful we should normally see about 30 -70% present.  Applications Applications of Gene Expression in Research and Development Applications of Gene Expression in Research and Development Expr