Download t - CBS

The Central Dogma
Life - a recipe for making proteins
Transcription
DNA
Translation
RNA
protein
TAAAAGAAATTAAAGAACAGCTTAAATAGCAGGACCGGGGATCAATCGGGG
AAAGTGTGAATAACTTTTCGGAAGTCATACACAGTCTGTCCACATGTGGATA
GGCTGTGTTTCCTGTCTTTTTCACAACTTATCCACAAATCCACAGGCCCTAC
TATTACTTCTACTATTTTTTATAAATATATATATTAATACATTATCCGTTAGGAG
GATAAAAATGAAATTCACGATTCAAAAAGATCGTCTTGTTGAAAGTGTCCAA
GATGTATTAAAAGCAGTTTCATCCAGAACCACGATTCCCATTCTGACTGGTA
TTAAAATTGTTGCATCAGATGATGGAGTATCCTTTACAGGGAGTGACTCAGA
TATTTCTATTGAATCCTTCATTCCAAAAGAAGAAGGAGATAAAGAAATCGTC
ACTATTGAACAGCCCGGAAGCATCGTTTTACAGGCTCGCTTTTTTAGTGAAA
TTGTAAAAAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTA
TTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGAT
GCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGA
TCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTC
CACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCA
AAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAG
GCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAA
AAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGA
TATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACGTCTTGTTC
TTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCTGATTCCGC
AAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCAT
TGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACA
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
Microarrays - The Technologies
Stanford Microarrays
Affymetrix
Why?
RNA
How?
gene
mRNA
gene specific DNA probes
labeled target
Stanford Microarrays
Stanford Microarrays
Coating glass slides
Deposition of probes
Post-processing
Hybridization
Coating
1. Rinse of slides:
NaOH and EtOH
2 h - shaking
2. Wash with water
3. Coat slides:
poly-L-lycine
1 h - shaking
4. Wash and dry
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
Microarrayer
Making Microarrays
1. Produce probes
• oligos
• cDNA library
• PCR products
2. Print by the use of a robot
3. Post-process:
• rehydrate
• snap dry
• UV-cross link
• block surface
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
Oligonucleotide Synthesis
NPPOC
O
NPPOC
B
P
CEO
NPPOC
O
B
HO
O
Detritylisation
O
(Deblock A solution)
B
O
Addition
O
CEO
CEO P O
O
(Amidite)
O
P
B
O
CEO P
O
N
NPPOC
B
O
DMTO
B
O
O
N
O
O
B
O
Photo-Deprotection
Oxidation
O
(Oxidizer)
B
O
(Deblock L)
Capping of uncoupled amidites
NPPOC
O
NPPOC
B
O
O
NPPOC
B
O
O
CEO P O
O
O
O
B
3
O
O
B
NPPOC
B
O
CEO P O
HO O
HC
O
O
O
B
NPPOC
B
O
CEO P O
O
O
O
O
B
B
O
CEO P O
O
O
O
CEO P O
O
O
B
O
O
O
B
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
NimbleExpress™ Array Program
- 282,000 unique features (probes)
- Design fee: 3,000 USD
- 800 USD/array (minimum 10)
- few weeks before delivery
- can be run on the Affymetrix equipment
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
Is it worth it?
Number of known positives
Known positives versus the total number of significantly affected
genes at 5 different cutoffs in the TnrA experiment
Qspline normalization
Linear normalization
Number of significantly affected genes
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
Break - 15 minuttes
After break:
- introduce yourself
- why are you here?