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Introduction to Microarrays
The Central Dogma
Life - a recipe for making proteins
DNA proteinRNATranslationTranscription
ATCTTTTTCGGCTTTTTTTAGTATCCACAGAGGTTATCGACAACATTTTCACATTACCAACCCCTGTGGACAAGGTTTTTTCAACAGGTTGTCCGCTTTGTGGATAAGATTGTGACAACCATTGCAAGCTCTCGTTTATTTTGGTATTATATTTGTGTTTTAACTCTTGATTACTAATCCTACCTTTCCTCTTTATCCACAAAGTGTGGATAAGTTGTGGATTGATTTCACACAGCTTGTGTAGAAGGTTGTCCACAAGTTGTGAAATTTGTCGAAAAGCTATTTATCTACTATATTATATGTTTTCAACATTTAATGTGTACGAATGGTAAGCGCCATTTGCTCTTTTTTTGTGTTCTATAACAGAGAAAGACGCCATTTTCTAAGAAAAGGAGGGACGTGCCGGAAGATGGAAAATATATTAGACCTGTGGAACCAAGCCCTTGCTCAAATCGAAAAAAAGTTGAGCAAACCGAGTTTTGAGACTTGGATGAAGTCAACCAAAGCCCACTCACTGCAAGGCGATACATTAACAATCACGGCTCCCAATGAATTTGCCAGAGACTGGCTGGAGTCCAGATACTTGCATCTGATTGCAGATACTATATATGAATTAACCGGGGAAGAATTGAGCATTAAGTTTGTCATTCCTCAAAATCAAGATGTTGAGGACTTTATGCCGAAACCGCAAGTCAAAAAAGCGGTCAAAGAAGATACATCTGATTTTCCTCAAAATATGCTCAATCCAAAATATACTTTTGATACTTTTGTCATCGGATCTGGAAACCGATTTGCACATGCTGCTTCCCTCGCAGTAGCGGAAGCGCCCGCGAAAGCTTACAACCCTTTATTTATCTATGGGGGCGTCGGCTTAGGGAAAACACACTTAATGCATGCGATCGGCCATTATGTAATAGATCATAATCCTTCTGCCAAAGTGGTTTATCTGTCTTCTGAGAAATTTACAAACGAATTCATCAACTCTATCCGAGATAATAAAGCCGTCGACTTCCGCAATCGCTATCGAAATGTTGATGTGCTTTTGATAGATGATATTCAATTTTTAGCGGGGAAAGAACAAACCCAGGAAGAATTTTTCCATACATTTAACACATTACACGAAGAAAGCAAACAAATCGTCATTTCAAGTGACCGGCCGCCAAAGGAAATTCCGACACTTGAAGACAGATTGCGCTCACGTTTTGAATGGGGACTTATTACAGATATCACACCGCCTGATCTAGAAACGAGAATTGCAATTTTAAGAAAAAAGGCCAAAGCAGAGGGCCTCGATATTCCGAACGAGGTTATGCTTTACATCGCGAATCAAATCGACAGCAATATTCGGGAACTCGAAGGAGCATTAATCAGAGTTGTCGCTTATTCATCTTTAATTAATAAAGATATTAATGCTGATCTGGCCGCTGAGGCGTTGAAAGATATTATTCCTTCCTCAAAACCGAAAGTCATTACGATAAAAGAAATTCAGAGGGTAGTAGGCCAGCAATTTAATATTAAACTCGAGGATTTCAAAGCAAAAAAACGGACAAAGTCAGTAGCTTTTCCGCGTCAAATCGCCATGTACTTATCAAGGGAAATGACTGATTCCTCTCTTCCTAAAATCGGTGAAGAGTTTGGAGGACGTGATCATACGACCGTTATTCATGCGCATGAAAAAATTTCAAAACTGCTGGCAGATGATGAACAGCTTCAGCAGCATGTAAAAGAAATTAAAGAACAGCTTAAATAGCAGGACCGGGGATCAATCGGGGAAAGTGTGAATAACTTTTCGGAAGTCATACACAGTCTGTCCACATGTGGATAGGCTGTGTTTCCTGTCTTTTTCACAACTTATCCACAAATCCACAGGCCCTACTATTACTTCTACTATTTTTTATAAATATATATATTAATACATTATCCGTTAGGAGGATAAAAATGAAATTCACGATTCAAAAAGATCGTCTTGTTGAAAGTGTCCAAGATGTATTAAAAGCAGTTTCATCCAGAACCACGATTCCCATTCTGACTGGTATTAAAATTGTTGCATCAGATGATGGAGTATCCTTTACAGGGAGTGACTCAGATATTTCTATTGAATCCTTCATTCCAAAAGAAGAAGGAGATAAAGAAATCGTCACTATTGAACAGCCCGGAAGCATCGTTTTACAGGCTCGCTTTTTTAGTGAAATTGTAAAAAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCCACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCCACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACACAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACATTGTGA
DNA
The Central Dogma
Hybridization
A
A
A
T
T
G
G
C
C
T
A
T
G
A
T
G
C
C
A
A
A
T
T
G
G
C
C
T
A
T
G
A
T
G
C
C
Introduction to Microarrays
Microarrays - The Concept
Measure the level of transcript from a
very large number of genes in one go
CELL
RNA
Microarrays - The Technologies
Stanford Microarrays
High-density
Why?
RNA
How?
gene specific DNA probeslabeled target
gene
mRNA
Stanford-type Microarrays
Stanford-type Microarrays
Coating glass slides
Deposition of probes
Post-processing
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
Microarrayer
Making Microarrays
1. Produce probes
2. Print by the use of a robot
• oligos• cDNA library• PCR products
3. Post-process:
• rehydrate• snap dry• UV-cross link• block surface
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
Catalog Arrays
HumanMouseRat ArabidopsisC. elegansCanineDrosophila
E. coliP. aeruginosaPlasmodium/AnophelesVitis vinifera (Grape) Xenopus laevisYeastZebrafish
NimbleExpress™ Array Program
Fluidic Station and Scanner
The Affymetrix Genechip®
TTT
T
T
TT
T
T
T
A
A
AA
A
A
A
AAA
Photolithographyin situ synthesis
Spacers bound to surface with photolabile protection groups
Mask #1Mask #2
Photolithography - Micromirrors
NimbleExpress™ Array Program
manufactured on Iceland by NimbleGen Systems Inc.
The Affymetrix GeneChip®
A gene is represented like this:
- Perfect Match (PM)- MisMatch (MM)
PMMM
PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT
NimbleGen Systems Inc.
~380.000 probes/array
They do most of the practical work
The Technologies - Flexibility
Stanford microarrays: Are flexible, but
new probes must be ordered each time
High-density: Are not flexible, ....unless you
order the NimbleExpress™ chip or use the
NimbleGen Systems
Analysis of Data
Normalization:Linear or non-linear
Is it worth it?
Nu
mb
er
of
kn
ow
n p
osit
ives
Number of significantly affected genes
Qspline normalization
Linear normalization
Known positives versus the total number of significantly affected genes at 5 different cutoffs in the
TnrA experiment
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