Introduction to Microarrays
The Central Dogma
The Central Dogma
Hybridization
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Introduction to Microarrays
Microarrays - The Concept
Measure the level of transcript from a
very large number of genes in one go
CELL
RNA
Why not Northern?
RNA
How?
gene specific DNA probeslabeled target
gene
mRNA
Microarrays - The Technologies
Stanford Microarrays
Affymetrix
Stanford Microarrays
Glass slides
Deposition of probes
Ready-to-use array
Hybridization
Making Microarrays
1. Produce probes
2. Print by the use of a robot
• oligos• cDNA library• PCR products
Spotting - Mechanical deposition of probes
16-pin microarrayer
Sample preparation
1. Design experimentQuestion?Replicates?Test?
2. Perform experiment
4. Label RNAAmplification?Direct or indirect?Label?
wild typemutant
3. Precipitate RNAEukaryote/prokaryote?Cell wall?
mRNAmRNA
cDNAcDNA
Cy3-cDNACy5-cDNA
SAMPLE CONTROL
Stanford
microarrays
DESIGN
and ORDER
PROBES
Affymetrix GeneChip® oligonucleotide array
• 11 to 20 oligonucleotide probes for each gene
• On-chip synthesis of 25 mers
• ~20.000 genes per chip
• good quality data – low variance
Example Catalog Arrays
HumanMouseRat ArabidopsisC. elegansCanineDrosophila
E. coliP. aeruginosaPlasmodium/AnophelesVitis vinifera (Grape) Xenopus laevisYeastZebrafish
NimbleExpress™ Array Program
Fluidic Station and Scanner
The Affymetrix Genechip®
TTT
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Photolithographyin situ synthesis
Spacers bound to surface with photolabile protection groups
Mask #1Mask #2
Photolithography - Micromirrors
NimbleExpress™ Array Program
The Affymetrix GeneChip®
A gene is represented like this:
- Perfect Match (PM)- MisMatch (MM)
PMMM
PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT
The Technologies
- Costs
- Flexibility
- Data Quality
Affymetrix
Spotter
Facility setup:Stanford Microarrays < 100,000 USDAffymetrix < 250,000 USD
The Technologies – Cost
Cost pr. array Stanford Microarrays 30-50 USDAffymetrix 300-400 USD
NimbleExpress™ Array Program - a bit more expensive
The Technologies - Flexibility
Stanford microarrays: Are flexible, but
new probes must be ordered each time
Affymetrix arrays: Are not flexible, unless
you order the NimbleExpress™ chip
The Technologies - Data Quality
Reproducibility of data:(Pearson’s correlation coefficient)
Stanford microarrays: 0.80 - 0.95
Affymetrix: 0.95
The Technologies - Choice of
Stanford microarrays: If you work with unsequenced speciesLow budget
Affymetrix: Only sequenced species High data quality
Analysis of Data
Normalization:Linear or non-linear
Statistical test:student’s t-testANalysis Of VAriance
(ANOVA)Analysis:
Principle Component Analysis (PCA) Clustering and visualization
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 Regulatory Network
ComparableGene Expression Data
Normalization
Image analysis
The DNA Array Analysis Pipeline
