Transcript Metabolitics Structural Genomic Protein States

Microarrays and Their Uses
Brad Windle, Ph.D.
628-1956
[email protected]
Profile
A set of data or characteristics pertaining to an item
Profiles are sometimes referred to as Signatures or Fingerprints
Cellular Profiles
Gene/Protein
Sequence
Protein
Structure
Protein
Expression
Gene
Expression
SNPs
Protein
States
Structural
Genomic
Cell State
Drug
Response
Drug
Structure
Methylation
Misc
Data
Metabolitics
Disease
Cellular Profiles
Gene/Protein
Sequence
Protein
Structure
Drug
Structure
Protein
Expression
Gene
Expression
SNPs
Protein
States
Structural
Genomic
Drug
Response
Methylation
Misc
Data
Metabolitics
Profiles Have Two Sides
Gene s / Samples Sample 1
Gene 1
1
Gene s / Samples Sample 1 Sample 2
GeneGene
21
1 5
2
Gene 2
5
3
3
23 23
65
GeneGene
3
Gene 4
2
1
Gene 4
2
Sample 2 Sample 3
2
3
Sample 3 Sample 4
33
17
32
17
22
23
65213
21
3
1
3
Sample 4
32
22
23
3
A gene profile across samples
and a sample profile across genes
Microarrays
and
Gene Expression Profiling
What do we want to know?
The smaller picture
What genes went up or down under an experimental condition?
The bigger picture
Are cells or tissues related based on the genes they express?
For an experimental cell model, are there conditions that
are similar based on changes in gene expression?
For certain experimental conditions, are there genes that
show similar patterns of change (co-regulated)?
How did we use to do this?
Probe for 1 gene
Analyze ~10 samples
Now with Microarrays
Analyzing 1 gene for 10 samples
Analyzing thousands of genes for 1 sample
Spotting Arrays
Affymetrix
cDNA synthesis labeling
Gene Expression Profiling
cell or condition
of interest
control or reference cell
hybridize to microarray
Applications of Gene Expression Profiling
Tissue or Tumor Classification
Gene Classification
Drug Classification
Drug Target Identification
Drug Response Prediction
Ross et al Nature Genetics 24:227 (2000)
The Color Map
Cells
12345abcde
Genes
1
2
3
4
a
d
c
d
Ross et al Nature Genetics 24:227 (2000)
Alizadeh et al. Nature 403:503 (2000)
Alizadeh et al. Nature 403:503 (2000)
Gene Expression Array
Genomic Content Array
Methylation Array (Chromatin Array)
Replication Array
SNP Array
Structural Genomic Profiling
Comparative Genomic Hybridization (CGH)
cell with losses or gains
hybridize to metaphase
chromosomes
normal cell
Genome Representation Profiling Using Arrays
Normal
Label DNA
Cancer
Label DNA
BAC or Oligo Array
CG H
Detection mainly for cancer and inherited
deletions
Tumor suppressor genes are deleted
Oncogenes are amplified
Methylation Profiling
me
CCGG
GGCC
me
PCR linkers
CCGG
GGCC
me
me
PCR Amplify / Label
Hpa II / PCR Amplify/ Label
hybridize to array
CpG Island Array
Profile cells based on methylation state
cell-type profiles
Differences in the methylated state of cancers
Compare methylation profiles to gene expression profiles
Replication Profiling
Synchronized cells in G1/S
time
BrdU
Labeling
cDNA or BAC Array
Profiling Transcription Factor-Interactive DNA
Immuno-precipitate w/
Ab to protein
Chromatin IP or ChIP
total genomic DNA
Combinatorial Elements Regulating Transcription
Single Nucleotide Polymorphism (SNPs) Profile
Personal fingerprint
Profile a small town
Figure out who’s related to who
What chromosomes came from whom
What regions came from whom
Figure out what meiotic exchanges occurred
Pharmacogenomics - SNPs that affect disease
and reaction to drugs
The ProfileData Sources
Gene expression
DNA microarrays, oligo or PCR, 20-30,000 genes
Structural genomics
DNA microarrays, BACs, ~one per 1Mb
Methylation
DNA microarrays, upstream sequences, CpG islands
SNPs
DNA microarray, oligos, millions of SNP sites
Protein expression
Ab microarray, 2D gels, chromatographics
Protein states
2D gels, <1000 proteins resolved
Drug response
brute force, 70,000 compounds screened
Metabolitics
chromatographics
DNA/protein sequence
Sequencing, <20 people sequenced, brute force
Drug structure
in silico
Protein structure
3D crystallography, NMR, brute force
Questions?