Transcript DNA microarray

DNA Microarray
Presented by: Akram Moslehi
High-throughput methods for
measuring cellular states
• Gene expression levels: RT-PCR, arrays
• Protein levels, modifications: mass spec
• Protein locations: fluorescent tagging
• Metabolite levels: NMR and mass spec
• Systematic phenotyping
Outline of the lecture
Overview of Microarray Technology
Applications
Types of Microarrays
Manufacturing
Instrumentation and Software
Data Analysis-Basic
What are Microarrays?
• Microarrays are simply small glass or silicon
slides upon the surface of which are arrayed
thousands of features (usually between 500 up to a
million)
• Using a conventional hybridization process, the
level of expression of genes is measured (for
instance)
• Microarrays are read using laser-based
fluorescence scanners
• The process is “high throughput”
Why use Microarrays?
•Determine what genes are active in a cell and
at what levels
• Compare the gene expression profiles of a
control vs treated
• Determine what genes have increased or
decreased in during an experimental condition
• Determine which genes have biological
significance in a system
• Discovery of new genes, pathways, and
cellular trafficking
Types of microarrays
• Spotted (cDNA) - by Patrick Brown in 1990
– Robotic transfer of cDNA clones or PCR products
– Spotting on nylon membranes or glass slides coated with poly-lysine
– ink-jet printing (Agilent)
• Synthetic (oligo) – by Stephen Fodor in 1991
– Direct oligo synthesis on solid microarray substrate
– Uses photolithography (Affymetrix) or ink-jet printing (Agilent)
• Labeling can be radioactive, fluorescent (one-color), or two-color
How do we manufacture a spotted microarray?
Spotted Glass Arrays
Uses cDNA, Oligonucleotide, protein, antibody
-Robotically spotted cDNAs or Oligonucleotides
-Printed on Nylon, Plastic, or Glass microscope slide
Agilent: Oligonucleotide Array
Glass slides characteristics
• excellent chemical resistance
against solvents,
•
good mechanical stability (increased
thermal strain point)
•
low intrinsic fluorescence properties.
Start with individual genes
Amplify
purity by sequencing or using on agarose gel and an
estimate of the DNA concentration
This is an important step because all the DNA
fragments should be of similar
concentration/molarity and size, to achieve
similar reaction kinetics for all hybridisations
“Spot” them on a medium, e.g. an ordinary glass
microscope slide that chemically modified glass slides
usually with poly(L-lysine) or other cross-linking chemical
coating materials such as polyethyleneimine polymer paminophenyl trimethoxysilane/diazotization
the DNA solution will be immobilised on the surface e.g.
covalent or non covalent. However in the course of poly
(L-lysine) the negatively charged phosphate groups in the
DNA molecule, form an ionic bond with the positively
charged amine-derivatised surface
steel
Spotted arrays
spotting pin
Spotting is done by a robot such as inkjet printing
1 nanolitre
spots
90-120 µm
diameter
concentration of
100-500 µg/ml
chemically modified slides
384 well source
plate
200-250µm
Longer sequence target:500-2000bp
Microarray Spotter
Robotic spotting
The post-print processing step :drying
of the DNA on the slide overnight at
room temperature and the use of UV
cross-linking to prevent subsequent
binding of DNA, and to decrease the
background signal upon hybridisation
of a labelled target
Cartridge-based Chips
-Miniaturized, high density arrays of DNA oligos within a plastic
housing
-One sample=One chip
(Affymetrix, Agilent, Applied Biosystems…)
-Uses single fluorescent dye
-More expensive
-Usually 20–25 bases in length
-10–20 different oligonucleotides for each gene
500,000 Probes
GeneChip Technology
Affymetrix Inc
• Miniaturized, high density arrays of 1,300,000 DNA
oligos 1-cm by 1-cm
• Manufacturing Process:
– Solid phase chemical synthesis and Photolithographic
fabrication techniques employed in semiconductor industry
– Oligonucleotides for each gene selected by computer
program to be the following:
Unique in genome
Nonoverlapping
Spotted Vs. Oligonucleotide array
Spotted Arrays
Affy Gene Chips
Relative cheap to make
(~$10 slide)
 Flexible - spot anything
you want (2Kbp)
 Cheap
so can repeat
experiments many times
 Highly
variable
spot
deposition
 Usually have to make your
own
 Cross hybridisation


Expensive ($500 or
more)
 Limited types avail, no
chance of specialized
chips
 Fewer repeated
experiments usually
 Increases specificity,
Decrease sensitivity
 Can buy off the shelf
Sample preparation and
labelling
(1) isolating a total RNA
containing m RNA
(2)converted into (cDNA)
(3)labelled with Cy3 , Cy5
(4)purified to remove
contaminants such as primers,
unincorporated nucleotides,
cellular proteins, lipids, and
carbohydrates
filter spin columns :Qiaquick
Sample preparation and
labelling
•
the sample preparation starts by isolating a total RNA
containing messenger RNA that ideally represents a
quantitative copy of genes expressed at the time of
sample collection (experimental sample & reference
sample).
• separately converted into complementary DNA (cDNA)
• each cDNA (Sample and Control) are labelled with a
different tracking molecule, often fluorescent cyanine
dyes (i.e. Cy3 and Cy5)
Array hybridisation
• the labelled cDNA (Sample and Control) are mixed
together
• purified to remove contaminants such as primers,
unincorporated nucleotides, cellular proteins, lipids, and
carbohydrates. Purification is usually carried out using
filter spin columns such as Qiaquick from Qiagen
• the mixed labelled cDNA is competitively hybridised
against denatured PCR product or cDNA molecules
spotted on a glass slide
(5) Before hybridisation, the microarray
slides are incubated at high temperature
with solutions of saline-sodium buffer
(SSC), Sodium Dodecyl Sulfate (SDS)
and bovine serum albumin (BSA) to
reduce background due to nonspecific
binding.
(6)The slides are washed after hybridisation,
first to remove any labelled cDNA that did not hybridise
on the array, and secondly to increase stringency of the
experiment to reduce cross hybridisation. The later is
achieved by either increasing the temperature or
lowering the ionic strength of the buffers.
Expression profiling with cDNA microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 1
Hybridization
Laser 2
Scanning
+
Analysis
Image Capture
Microarray
confocal scanner
Image analysis of cDNA array
Expression profiling with DNA
microarrays Data acquistion
cDNA “control”
Cy3 labeled
1
2
2
5
3
6
5
2
5
2
5
cDNA “treament”
Cy5 labeled
1
6
6
3
5 4
3
6
Relative
intensity Output
HYBRIDIZATION
1
1
2
2
Spot #1
2
2
3
3
3
Spot #2
Spot #3 33
Spot #4
4
5
5
5
5
5 5
5 5
6
6
6 6
6 6
Spot #5
Spot #6
5
5
6 5
Relative
intensity
6
Output
Reading an array (cont.)
Block
Column
Row
Gene Name
Red
Green
Red:Green
Ratio
1
1
1
tub1
2,345
2,467
0.95
1
1
2
tub2
3,589
2,158
1.66
1
1
3
sec1
4,109
1,469
2.80
1
1
4
sec2
1,500
3,589
0.42
1
1
5
sec3
1,246
1,258
0.99
1
1
6
act1
1,937
2,104
0.92
1
1
7
act2
2,561
1,562
1.64
1
1
8
fus1
2,962
3,012
0.98
1
1
9
idp2
3,585
1,209
2.97
1
1
10
idp1
2,796
1,005
2.78
1
1
11
idh1
2,170
4,245
0.51
1
1
12
idh2
1,896
2,996
0.63
1
1
13
erd1
1,023
3,354
0.31
Campbell & Heyer, 2003
1
1
14
erd2
1,698
2,896
0.59
34
Image Analysis & Data
Visualization
Cy3
Cy5
Cy5
Cy3
200 10000 50.00
4800 4800 1.00
9000 300 0.03
Underexpressed
Genes
Experiments
Overexpressed
35
Cy5
log2 Cy3
5.64
0.00
-4.91
8
4
2
fold
2
4
8
Normal vs. Normal
Normal vs. Tumor
All configurations assume the DNA on the
array is in excess of the hybridized sample,
thus the kinetics are linear and the spot
intensity reflects that amount of hybridized
sample.
Validating Microarray Expression Data
Microarray data are not stand alone results and
requires validation by second method
Microarray data is only semi-quantitative
because of a limited dynamic range.
True quantitative results must be determined
with another technique such as Quantitative
real-time PCR
Microarray Validation
Two types of validation
1] Validating the instrument data using the same RNA (confirming a result)
And most importantly
2] Validating the biological phenomenon with new samples same
experiment conditions
Methods
Northern Blots, Immunohistochemistry, Western Blot,
PCR- i.e.Quantitative real-time PCR
**DNA mapping Arrays or CGH may also help indicate where or why a
change is occuring
Microarray Future
Diagnostics -[Affy, Nanogene only at this time]
– Disease detection
– Tumor classification
– Patient stratification
– Intervention therapeutics
Treatment and Customized Medicine
Clinical arrays currently available are the AmpliChip
CYP450 by Affymetrix and Roche. Used for predictive
phenotyping in defects of the cytochrome P450 Genes
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