Download DNA Microarray Experiments and Analysis

DNA Microarray Experiments and Analysis
John Wyrick
MBios 503
12/6/2004
mRNA level
Down
Up
Mechanics of DNA Microarrays
complex mixture of
mRNA molecules
Fluorescent
Dye
Gene 1
Gene 2
UACGGCUUAGCAGGUCAUUGG
ATGCCGAATCGTCCAGTAACC
...Gene 6200
Constructing DNA Microarrays
§ A DNA microarray is a collection of DNA probes separated in a regular
array atop a solid support (glass slide, silicon chip, etc)
§ Affymetrix oligonucleotide microarrays
• Short oligonucleotide probes (25mers) are synthesized directly on
a silicon wafer using photolithography methods
• Multiple oligonucleotide probes per gene including mismatch probe
controls
§ cDNA microarrays
• Full length cDNAs are PCR amplified and then printed using a robot
onto coated glass slides
• Glass slides can be coated with poly-lysine, GAPS, etc to facilitate
attachment of DNA to the glass slide
§ Oligonucleotide microarrays
• Long (70 - 100mers) oligonucleotide probes are printed on coated
glass slides
cDNA Microarray
For Yeast:
• Array of 6361 spots, each
representing a single yeast
gene
• Printed on GAPS-coated glass
microscope slide
• Each spot contains 500 pg
of PCR product
• Size of PCR products range
from 60 bp to 1500 bp;
average size: 480 bp
Affymetrix Oligonucleotide Microarrays
• 6181 ORFs + alignment controls
• each ORF: 20 perfect match 25mers (PM)
20 single base mismatch (MM)
• mRNA signal = average of differences
(PM - MM)
• Sensitivity = 0.1 mRNA molecules/cell
• Dynamic range = 0.1 - 100 mRNAs/cell
• Reproducibility = wt vs wt experiment, 99%
of genes within 1.7 fold
The Mechanics of a DNA Microarray Experiment
§ Isolate mRNA from cell cultures
§ Reverse Transcribe mRNA into cDNA
§ Label cDNA or cRNA by incorporating fluorescently-labeled nucleotides
§ Hybridize labeled cDNA to DNA microarray
§ Wash and scan microarray in confocal laser scanner
§ Analyze data
DNA Microarray Experimental Procedure
Extracting Data from Scanned Microarray Image
Scanned Image
Gene with altered mRNA levels
§ Grid
• The total amount of fluorescence
intensity of Cy5 and Cy3 dyes
inside each grid circle is calculated
• Grid can be aligned manually or
automatically
Experimental mRNA (Cy5)
Control mRNA (Cy3)
Merge
Initial Microarray Data Analysis
§ Data Normalization
• Total signal normalization
• Exogenous (spiked) control mRNAs
§ Filters for identifying genes with altered mRNA levels
• Absolute intensity change thresholds
• Fold-change cutoffs
• T-tests and other significance tests
• Permutation tests
• Error model methods
§ Use a Database to normalize, filter, and analyze
microarray data
• Integrate microarray data with data from other
sources (gene function, proteomics data, etc)
Microarray Database
Literature
-Gene annotations
Results
Analysis Tools
Raw data
DataBase
-Microarray data
-mySQL
Perl
Perl
Web interface
-Error model analysis
-Cluster analysis
-Chromosome display
Methods for Data Mining and Visualization
§ Data visualization tools
• Displays microarray data using prior knowledge of gene location/function
§ Clustering
• Simplifies analysis by grouping together genes with similar expression
patterns, but does not infer relationships between genes
§ Boolean Comparisons
• Compares different microarray data sets to identify significant overlaps
in expression patterns
§ Hidden Markov models
• Useful for clustering time course data
§ Bayesian networks
• Useful for identifying and testing relationships between genes
Raw Microarray Data
GCN5 deletion
Gene
ORF YAL069W
SEO1 (YAL067C)
ORF YAL066W
ORF YAL065C
ORF YAL065C-A
ORF YAL063C
GDH3 (YAL062W)
ORF YAL061W
ORF YAL060W
SIM1 (YAL059W)
CNE1 (YAL058W)
ORF YAL058C-A
ORF YAL056W
ORF YAL055W
ACS1 (YAL054C)
ORF YAL053W
ORF YAL051W
ORF YAL049C
ORF YAL048C
ORF YAL047C
ORF YAL046C
ORF YAL045C
GCV3 (YAL044C)
PTA1 (YAL043C)
ORF YAL042W
ORF YAL043C-A
CDC24 (YAL041W)
CLN3 (YAL040C)
CYC3 (YAL039C)
CDC19 (YAL038W)
Gene (parse)
YAL069W
YAL067C
YAL066W
YAL065C
YAL065CA
YAL063C
YAL062W
YAL061W
YAL060W
YAL059W
YAL058W
YAL058CA
YAL056W
YAL055W
YAL054C
YAL053W
YAL051W
YAL049C
YAL048C
YAL047C
YAL046C
YAL045C
YAL044C
YAL043C
YAL042W
YAL043CA
YAL041W
YAL040C
YAL039C
YAL038W
WT1val
2.0
13.0
10.0
10.0
21.0
9.0
17.0
21.0
96.0
182.0
56.0
12.0
52.0
43.0
15.0
51.0
8.0
117.0
5.0
0.0
44.0
2.0
339.0
23.0
167.0
10.0
1.0
37.0
86.0
2651.0
WT1call
A
A
A
A
A
A
M
P
P
P
P
A
P
P
A
P
A
P
A
A
P
A
P
P
P
A
A
P
P
P
MT1val
6.9
6.9
2.5
6.3
3.1
0.6
11.3
12.0
53.5
105.7
33.3
23.3
56.6
29.6
0.6
52.2
16.4
112.6
17.6
12.6
18.9
12.6
315.8
43.4
118.3
12.0
110.1
70.5
78.6
2257.6
MT1call
A
A
A
A
A
A
A
A
A
P
P
A
P
A
A
P
A
P
A
A
A
A
P
P
P
A
P
P
P
P
WT2val
0.7
11.3
20.0
7.3
14.0
20.0
18.0
16.0
58.1
154.2
40.7
0.7
66.8
13.4
6.0
61.4
22.7
98.8
7.3
12.0
40.7
7.3
236.3
41.4
140.2
5.3
12.7
50.7
72.1
3955.4
WT2call
A
A
P
A
A
A
M
A
P
P
P
A
P
A
M
P
P
P
A
A
P
A
P
P
P
A
A
P
P
P
MT2val
0.0
0.9
16.4
2.7
23.7
2.7
3.6
9.1
57.4
96.6
29.2
25.5
67.5
65.6
0.0
95.7
8.2
101.2
0.9
5.5
29.2
5.5
255.2
50.1
97.5
20.1
24.6
34.6
58.3
4586.3
MT2call
A
A
A
A
A
A
A
A
P
P
M
A
P
P
A
P
A
P
A
A
A
A
P
P
P
A
A
P
P
P