Download OverviewJan06

Introduction to
Microarrays
The Central Dogma
Life - a recipe for making proteins
Transcription
DNA
Translation
RNA
protein
GCTTTTTTAGTGAAATTGTAAAAAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAA
AATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGAT
GCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCCCAA
CTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCCACCTCAGAAACA
CGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAAGTGAATTATTATGCACTG
CAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACTTGATATTCCAGAAGACAG
ATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATG
ACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAA
AAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCT
GATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCC
ATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAATTGCCGATGGCAACTG
TAGAAATTGAAGTCCAAAATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTA
ATCTAAACGGACTGGATGCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCA
TGCGATTCAGATCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAG
TGTCCACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAA
GTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACT
TGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAAC
TCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAA
GTTCTGTTTAAAGCGAAAAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATC
CAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAA
AAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAC
AGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAA
AGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAA
GAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACATTG
TGA
DNA
The Central Dogma
Hybridization
A
T
A T
G
C
G C
C
G
C G
A
T
A T
C
G
C G
T
A
T A
G
C
G C
T
A
T A
A
T
A T
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 Northen?
RNA
How?
gene
mRNA
gene specific DNA probes
labeled target
The DNA Array Analysis Pipeline
Question
Experimental Design
Array design
Probe design
Sample Preparation
Hybridization
Buy Chip/Array
Image analysis
Normalization
Expression Index
Calculation
Comparable
Gene Expression Data
Statistical Analysis
Fit to Model (time series)
Advanced Data Analysis
Clustering
PCA
Classification
Promoter Analysis
Regulatory Network
Microarrays - The Technologies
Stanford Microarrays
Affymetrix
Stanford Microarrays
Glass slides
Deposition of probes
Post-processing
Hybridization
Making Microarrays
1. Produce probes
• oligos
• cDNA library
• PCR products
2. Print by the use of a robot
Spotting
- Mechanical deposition of probes
16-pin microarrayer
Making Microarrays
1. Produce probes
• oligos
• cDNA library
• PCR products
2. Print by the use of a robot
3. Post-process:
Sample preparation
1. Design experiment
Question?
Replicates?
Test?
mutant
wild type
2. Perform experiment
3. Precipitate RNA
4. Label RNA
Eukaryote/prokaryote?
Cell wall?
Amplification?
Direct or indirect?
Label?
Stanford microarrays
CONTROL
SAMPLE
mRNA
mRNA
DESIGN
and ORDER
cDNA
Cy5-cDNA
PROBES
cDNA
Cy3-cDNA
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
Catalog Arrays
Human
Mouse
Rat
Arabidopsis
C. elegans
Canine
Drosophila
E. coli
P. aeruginosa
Plasmodium/Anopheles
Vitis vinifera (Grape)
Xenopus laevis
Yeast
Zebrafish
NimbleExpress™ Array Program
Fluidic Station and Scanner
The Affymetrix Genechip®
Photolithography
in situ synthesis
T
A
T
T
T
A
A
T
Mask #2
Mask #1
T
A
A
T T T
A
T
A
AA A
Spacers bound to surface with
photolabile protection groups
Photolithography - Micromirrors
NimbleExpress™ Array Program
The Affymetrix GeneChip®
A gene is represented like this:
PM
MM
- Perfect Match (PM)
- MisMatch (MM)
PM: CGATCAATTGCACTATGTCATTTCT
MM: CGATCAATTGCAGTATGTCATTTCT
The Technologies
- Costs
Spotter
- Flexibility
- Data Quality
Affymetrix
The Technologies - Cost
Facility setup:
Stanford Microarrays
Affymetrix
< 100,000 USD
< 250,000 USD
Cost pr. array
Stanford Microarrays
Affymetrix
30-50 USD
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 species
Low budget
Affymetrix:
Only sequenced species
High data quality
Analysis of Data
Normalization:
Linear or non-linear
Statistical test:
student’s t-test
ANalysis Of VAriance (ANOVA)
Analysis:
Principle Component Analysis (PCA)
Clustering and visualization