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19a microarrays

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  • 1. Microarrays Microarrays are used to examined thousands of mRNA strands all at the same time. Two samples: 1. Control (normal / wild type) 2. Treatment (e.g. chemical) / Abnormal (e.g. cancer)
  • 2. Microarrays  Up to 100 000 pieces of known DNA sequence are placed onto the slide  The position of each DNA sequence is known and kept in a database
  • 3. Microarrays
  • 4. Steps to Microarray Analysis 1. mRNA is isolated from tissue 2. cDNA is made using mRNA as template (using reverse transcriptase) cDNA will also be labeled with a fluorescent protein   e.g. control = green; drug treatment = red 3. fluorescent cDNA is placed onto the microarray 4. laser scanner to locate fluorescence of each sample
  • 5. cDNA Microarray Assay estrogen in sesame oil (vehicle) sesame oil harvest tissue extract RNA Incorporate Cy3 label Incorporate Cy5 label Mix and hybridize Scan Data Collection Data analysis
  • 6. Microarray Animation
  • 7. Scanned Microarray
  • 8. Scanned Microarray 1. Green  more mRNA produced in control sample 2. Red  more mRNA produced in treated sample 3. Yellow  both control and treated sample produce the same amount of mRNA
  • 9. Microarray Information What do you do with all these colours? Focus on the green or red dots  these represent mRNA that are different between the control and the test sample If you know which gene is being changed, you may understand which metabolic pathway is being affected.