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INTRODUCTIONThe Technology of Microarrays
January 2009 - Hanne Jarmer
The Concept
gene specific DNA probes labeled target
gene
mRNA
Spotted arrays
High-density arrays
5 - 11 micron features
13 -16 micron features ~60 micron features
The spotting
Coating glass slides
Deposition of probes
Post-processing
Hybridization
The spotting
mRNAmRNA
cDNAcDNA
Cy3-cDNACy5-cDNA
SAMPLE CONTROL
Spotted microarrays
mRNAmRNA
cDNAcDNA
Cy3-cDNACy5-cDNA
SAMPLE CONTROL
Spotted microarrays
The Output
Cy5 Cy3
Affymetrix GeneChip® oligonucleotide array
• 11 to 20 oligonucleotide probes for each gene• Expensive to make• On-chip synthesis of 25 mers• ~20,000 genes per chip• High-quality data• Up to 6.4 M features
Affymetrix GeneChip® oligonucleotide array
The Chip
5 - 11 micron features
Photolithographyin situ synthesis
Spacers bound to surface with photolabile protection groups
Photolithographyin situ synthesis
Spacers bound to surface with photolabile protection groups
TTT
Photolithographyin situ synthesis
TTT AAA
Photolithographyin situ synthesis
The Eberwine Protocol
The Eberwine ProtocolSAMPLE
The Eberwine Protocol
RNA
SAMPLE
The Eberwine Protocol
RNA
T7
SAMPLE
70 °C10 min
The Eberwine Protocol
RNA
T7
SAMPLE
ssDNA
+ Reverse Transcriptase
42 °C2 h
70 °C10 min
The Eberwine Protocol
RNA
T7
dsDNA
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase
42 °C2 h
16 °C2 h
70 °C10 min
The Eberwine Protocol
RNA
T7
dsDNA
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase clean up dsDNA
42 °C2 h
16 °C2 h
70 °C10 min
The Eberwine Protocol
RNA
T7
dsDNA
T7 pol
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase
42 °C2 h
16 °C2 h
70 °C10 min
The Eberwine Protocol
RNA
T7
dsDNA
T7 pol
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase
+ Biotin-labeled nucleotides
42 °C2 h
16 °C2 h
37 °C6 h
70 °C10 min
The Eberwine Protocol
RNA
T7
dsDNA
T7 pol
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase
+ Biotin-labeled nucleotides
aRNA
42 °C2 h
16 °C2 h
37 °C6 h
70 °C10 min
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
biotinylated anti-anti IgG
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
biotinylated anti-anti IgG
Each gene is represented like this:
- Perfect Match (PM)- MisMatch (MM)
PMMM
CGATCAATTGCACTATGTCATTTCT CGATCAATTGCAGTATGTCATTTCT
PM:MM:
Roche-NimbleGen
Roche-NimbleGen
• High density: 70,000 to 2.1 M features
• Low design costs
• Probe length: 20-70 mers
• Multi-well system
• Full service!
Photolithography - micromirrors
13 - 16 micron features
Agilent
Agilent
• Density: 15-244 K features
• No design fee
• Probe length: 20-70 mers
The output
Cyanine5
Overlay
Cyanine3
Ink-Jet Printing
< 1 msec
Resistor on/off - vapourizing liquid
Multi-well formats
• Low Density, Low Cost• Whole/Partial Genome• Single Probe Per Gene• 4 x 44K • 8 x 15K
• Medium Density• Premium Partial Genome • Replicate Features• 2 x 105K
• High Density• Premium Whole Genome • Replicate Features• High Performance 244K
• Gene expression• SNP analysis• ChIP-chip (detection of TF-binding sites)• Comparative Genomic Hybridization (CGH)• Whole genome tiling arrays
Applications
Comparative Genomic Hybridization
Comparing two genomes
ChIP-chip
ChIP = Chromatin Immuno Precipitation
SNP analysisPatients
Controls
Association study
Sample PreparationHybridization
Array designProbe design
QuestionExperimental Design
Buy Chip/Array
Statistical AnalysisFit to Model (time series)
Expression IndexCalculation
Advanced Data AnalysisClustering PCA Classification Promoter Analysis
Meta analysis Survival analysis Regulatory Network
ComparableGene Expression Data
Normalization
Image analysis
The DNA Array Analysis Pipeline
Coffee BreakBe back at 10:30
