1. Microarrays: Basic Principle AGCCTAGCCT ACCGAACCGA GCGGAGCGGA CCGGACCGGA TCGGATCGGA Probe Targets Highly parallel molecular search and sort process based on hybridization of nucleic acids

2. Probe ArrayTargets Mix Probes and Target: Selective Hybridization Microarray Principles

3. Probe ArrayTargets Microarray Principles ~10 6 probes per spot

4. Microarray Principles Probe Array Gene ExpressionLevel Gene1: 1846 Gene2: 368 ………….…….. Gene10,000: 218

5. Synthesized High Density Oligonucleotide Chip Design Gene sequence database Design a set of short probes for each gene 5’ 3’ 5’ 3’ Optimize probe choices with BLAST Synthesize probes ACGTAC and controls on the array ACCTAC

6. GATCCTGACTCTTCTTCAC ….. GAGAAG Z20656 probe Sequence databaseProbe for the alpha-myosin gene sequence GAGAAG Synthesize probe on chip (a) (b) probe Software to ensure probes are specific to intended target sequences #!/usr/bin/perl $x= $x=~s/\/.+?\n/g; Print OutputFile1 “$x”; Sequence database Probes for all sequences Optimize Synthesize probes Oligonucleotide Array Technology: Chip Design

7. RNA Extraction, Amplification, Labeling Tissue Total RNA cDNA (from mRNA only) R-TranscriptionTranscriptionAmplification c mRNA (biotin labeled) Biotin labeled bases Oligo-dT Fragment, Hybridize, Stain Laser scan

8. Microarray Processed Output Gene Level Gene1: 1846 Gene2: 368 ………….…….. Gene22,283: 218

9. cRNA from tissue of interest DNA microarray Hybridization Laser scanning Image Data processing Quantitative gene expression (a) (b) Multiple experimentsPattern discovery Combine, normalize, analyze Oligonucleotide Array Technology: Experimental Approach