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What Is Microarray. A new powerful technology for biological exploration Parallel High-throughput Large-scale Genomic scale. Microarray. Replication. Transcription. Translation. DNA. RNA. Protein. Reverse transcription. DNA. ATATCGGCATCAGTCGATCGATCATCGATCGAT. mRNA.
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What Is Microarray • A new powerful technology for biological exploration • Parallel • High-throughput • Large-scale • Genomic scale
Replication Transcription Translation DNA RNA Protein Reverse transcription DNA ATATCGGCATCAGTCGATCGATCATCGATCGAT mRNA UAUAGCCGUAGUCAGCUAGCUAGUAGCUAGCUA cDNA ATATCGGCATCAGTCGATCGATCATCGATCGAT
What Is Microarray • Different Approaches
Affymetrix • Probe Array (Photolithography) • Synthesis of probe • Hybridization
Stanford Approach • Use robot to spot glass slides • Able to measure qualitatively relative expression levels of genes • Differential expression by use of simultaneous, two-color fluorescence hybridisation • Cheaper with DIY ($60,000) • Also called home-made system
Life Cycle(cont.) Cy3 (green) Cy5 (red)
Arrayer Hardware Scanner Software Procedures • Preparation • Target DNA (reference and test samples) • Slides • Reaction(Droplet or Pin Spotting) • Hybridization • Scanning • Analysis • Image processing • Data mining • Modeling
Instruments • Arrayer • Scanner • Laser Confocal Microscope
Scanner • GenPix 4000
Problems – by direct view Anti-probe Locally high background Spot overlap Precipitate Locally low signal Comet-tails (donut hole)
Problems –related wit IP • Local problems • Spot position variation • Spot shape and size irregularity • Spot Overlapping • Global problems • High background • Low signal • White-noise/Speckle effect (Contamination)
Array Alignment Good Alignment Bad Alignment
Spot Finding • Grid System Determination • Manual • Semiautomatic • Automatic • Spot Segmentation • Space-based • Intensity-based • Frequency-based • Hybrid approach
Applications • Gene expression studies • Gene function for cell state change in various conditions • Disease Diagnosis • Pathogen Analysis
Applications(Cont.) • Drug Discovery • identify appropriate molecular targets for therapeutic intervention • monitor changes in gene expression in response to drug treatments • Trageted Drug Treatment
….. Gene index 1.75 Log(R/G) …..
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