Microarrays The Next Revolution Dana Arico Betsy Karle Jeff Kucharski Kumo Lartevi Dr' Terrie Rife

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Microarrays The Next Revolution Dana
AricoBetsy Karle Jeff Kucharski Kumo
LarteviDr. Terrie Rife
To Me Make sure students take pretest first!!!!
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Microarray Background

• Allows researchers to analyze the relative gene
  expression of thousands of genes at one time.
• DNA -----------------------gt
  RNA

Transcriptional Regulation

• Complete sequencing of model organisms has made
  this technique possible
• We know the sequence of most but not all RNAs in
  the sequenced genomes!

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1. Make an Arrayed Southern Blot Membrane in
Miniature
Genome
Cloning Sequencing
cDNA
Robotic Printing
Arrayed Genes
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Making the Probes
Non-alcoholics Alcoholics
Extraction
RNA
RT reaction
cDNA Labeling
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Microarray Printer
UV Cross-Link when finished
http//150.135.37.182/dgalbrai/robot/
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A Microarray Experiment

• Frontal Cortex of
• Alcoholics vs. Non - Alcoholics
• How many genes do you expect to change in
  expression in this case?
• By how much do you expect their expression to
  change?
• Can we find out how much of these genes are in
  the brain?
• Can we answer these questions using a microarray?

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The Whole Picture
Labeled cDNA
Arrayed Genes
Hybridization
Hybridized Array
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Gene Expression
Non-Alcoholics Alcoholics 1) I
1) 2) II 2) II 3) 3) I
I
I
II II
I
I
I
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Thousands of Genes
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- 4 of 4,000
RNAs
Tested Were Different
Between Alcoholics
And Non-Alcoholics by 40 or More
Quantify???
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Uses of Microarrays
Many Applications of Microarrays Examples 1)
Gene Expression Studies 2) Mutations/ Single
Nucleotide Polymorphisms 3)Disease
Diagnosis 4) What drug should we treat with?
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Another Way to Make Arrays- need to do SNP
Analsysis
http//www.bio.davidson.edu/courses/Molbio/MolStud
ents/spring2000/hillmann/Microarrays.html
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What are we going to do? Dr. Slekar - Studying
Oxidative Stress Dr. Daniel - Studying Glucose
Transport

• Harvest RNA from Yeast
• Wild- Type
• Yeast that have an over-expressed gene
• Or Yeast Lacking Hexose Transporters
• Compare their Transcription

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Working with Yeast
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mRNA strand
3_____________________
To Dr. Malcolm Campbell
Note Web Animation
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Chip Reader
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.tiff data
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Scanalyze/gridding
 
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Genespring
Ratio Mutant Expression / Wildtype expression
Red Mutant gene overexpressed Blue- Mutant gene
underexpressed Grey- No readable
data www.sigenetics.com
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Genome of Yeast PHO 85 knockout
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Cell Cycle control
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Gene FilteringExpression Ratios less then 0.1
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Cluster Analysis
1. Allows Identification of genes that
participate in various Biological processes -
groups genes 2. Find Clues about the function of
previously unknown genes
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Info from http//online.itp.ucsb.edu/online/infobi
o01/domany1/
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Considerations to improve the Results of a
Microarray Experiment

• Replicate each experiment on multiple arrays
• Dye reversal
• Randomize the address of arrayed DNA
• Large number of controls
• Intensity series clones
• Background hybridization control
• Contamination controls

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(green mutant/ red WT)
Three Pho85D Microarrays
(red mutant/green WT)
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2 Experiments-- Different Results
Red Mutant/ Green WT
Green Mutant/ Red WT