Microarray Experimental Set Up

Department of Statistics
University of Pune
Pune - 411007

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically
Glass (sometimes of other material like nylon) slide on which
single stranded DNA molecules are attached at fixed positions
called as spots

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically
Glass (sometimes of other material like nylon) slide on which
single stranded DNA molecules are attached at fixed positions
called as spots
Array sizes: Microscope slide (2.5cm × 7.5cm)

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically
Glass (sometimes of other material like nylon) slide on which
single stranded DNA molecules are attached at fixed positions
called as spots
Array sizes: Microscope slide (2.5cm × 7.5cm)
Square silicon chips (0.5cm × 0.5cm)

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically
Glass (sometimes of other material like nylon) slide on which
single stranded DNA molecules are attached at fixed positions
called as spots
Array sizes: Microscope slide (2.5cm × 7.5cm)
Square silicon chips (0.5cm × 0.5cm)
Thousands of spots on an array, each containing a number of
identical DNA molecules or fragments of identical molecules

Microarray Experimental Set Up

Microarray Experimental Set up

Microarray experiment: Innovative technology which allows

monitoring of expression levels for thousands of genes
simultaneously
Microarray is a solid base on which a grid of spots or droplets
of genetic material of known sequence is arranged
systematically
Glass (sometimes of other material like nylon) slide on which
single stranded DNA molecules are attached at fixed positions
called as spots
Array sizes: Microscope slide (2.5cm × 7.5cm)
Square silicon chips (0.5cm × 0.5cm)
Thousands of spots on an array, each containing a number of
identical DNA molecules or fragments of identical molecules
Spot diameter: Of order of 0.1 mm or even smaller
Microarray Experimental Set Up
Microarray Experimental Set Up
Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.
Two approaches of microarray fabrication: cDNA and
oligonucelotide microarray

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.
Two approaches of microarray fabrication: cDNA and
oligonucelotide microarray
cDNA microarray

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.
Two approaches of microarray fabrication: cDNA and
oligonucelotide microarray
cDNA microarray
mRNA is usually unstable and subject to degradation

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.
Two approaches of microarray fabrication: cDNA and
oligonucelotide microarray
cDNA microarray
mRNA is usually unstable and subject to degradation
Using mRNA as a template a complementary DNA strand is
synthesized using reverse transcription

Microarray Experimental Set Up

Every spot on the array represents an independent
experimental assay for the presence and abundance of a
specific sequence of bases in the sample polynucleotide strand
Making and using printed DNA microarrays require two pieces
of hardware, an arrayer to print the spots on the array and a
scanner to image the hybridized array.
Two approaches of microarray fabrication: cDNA and
oligonucelotide microarray
cDNA microarray
mRNA is usually unstable and subject to degradation
Using mRNA as a template a complementary DNA strand is
synthesized using reverse transcription
Complementary DNA thus formed is known as cDNA

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence
cDNA is generally of length 500 to 5000 bases of known
sequence

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence
cDNA is generally of length 500 to 5000 bases of known
sequence
Oligonucelotide microarray

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence
cDNA is generally of length 500 to 5000 bases of known
sequence
Oligonucelotide microarray
Oligonucleotides (oligo for short) are short sequential
base-pair segments, ranging from 15 to 70 nucleotides in
length, taken from hundreds of nucleotides in a DNA segment
that functions as a gene

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence
cDNA is generally of length 500 to 5000 bases of known
sequence
Oligonucelotide microarray
Oligonucleotides (oligo for short) are short sequential
base-pair segments, ranging from 15 to 70 nucleotides in
length, taken from hundreds of nucleotides in a DNA segment
that functions as a gene
On cDNA microarray cDNAs are printed

Microarray Experimental Set Up

Labeled as cDNA because its sequence is complementary to
the original mRNA sequence.
It is stable and not easily degraded
Single stranded cDNA molecule is shorter than the parent
DNA sequence, as it will have only its coding exon sequence
cDNA is generally of length 500 to 5000 bases of known
sequence
Oligonucelotide microarray
Oligonucleotides (oligo for short) are short sequential
base-pair segments, ranging from 15 to 70 nucleotides in
length, taken from hundreds of nucleotides in a DNA segment
that functions as a gene
On cDNA microarray cDNAs are printed
On spotted oligonucleotide arrays oligos are printed
Microarray Experimental Set Up
Microarray experiment consists of following stages
(i) Preparation of probe DNA.
(ii) Printing of DNA.
(iii) Post processing of slides.
(iv) Preparation of target.
(v) Hybridization.
(vi) Slide imaging and data collection.
Knowledge of experiment set up is essential to understand the
variability in the data generated by the microarray experiments

Microarray Experimental Set Up

Preparation of probe DNA: For organisms whose genomes
have been fully sequenced, every known open reading frame in
the genome is amplified and used as a probe

Microarray Experimental Set Up

Preparation of probe DNA: For organisms whose genomes
have been fully sequenced, every known open reading frame in
the genome is amplified and used as a probe
Amplification is carried by a PCR amplification technique

Microarray Experimental Set Up

Preparation of probe DNA: For organisms whose genomes
have been fully sequenced, every known open reading frame in
the genome is amplified and used as a probe
Amplification is carried by a PCR amplification technique
It allows multiplication of DNA fragments by millions in a few
hours. This provides sufficient DNA for printing many arrays

Microarray Experimental Set Up

Preparation of probe DNA: For organisms whose genomes
have been fully sequenced, every known open reading frame in
the genome is amplified and used as a probe
Amplification is carried by a PCR amplification technique
It allows multiplication of DNA fragments by millions in a few
hours. This provides sufficient DNA for printing many arrays
ESTs can be used to identify distinct mRNA transcripts, and
individual cDNA clones corresponding to each of these
transcripts can be used as the source of gene-specific probes
on an array

Microarray Experimental Set Up

Preparation of probe DNA: For organisms whose genomes
have been fully sequenced, every known open reading frame in
the genome is amplified and used as a probe
Amplification is carried by a PCR amplification technique
It allows multiplication of DNA fragments by millions in a few
hours. This provides sufficient DNA for printing many arrays
ESTs can be used to identify distinct mRNA transcripts, and
individual cDNA clones corresponding to each of these
transcripts can be used as the source of gene-specific probes
on an array
The basic task in DNA printing is to make the final DNA
solution to be as free from contamination as possible and also
from detergents, glycerol and other particulate matter which
interfere at numerous stages in printing process

Microarray Experimental Set Up

Printing of cDNA array

Microarray Experimental Set Up

Printing of cDNA array
The cDNAs are printed on Poly-L-lysine coated glass
microscope slides

Microarray Experimental Set Up

Printing of cDNA array
The cDNAs are printed on Poly-L-lysine coated glass
microscope slides
The arraying robot is used in printing cDNA microarray

Microarray Experimental Set Up

Printing of cDNA array
The cDNAs are printed on Poly-L-lysine coated glass
microscope slides
The arraying robot is used in printing cDNA microarray
Large number of slides are placed on and secured to a platter

Microarray Experimental Set Up

Printing of cDNA array
The cDNAs are printed on Poly-L-lysine coated glass
microscope slides
The arraying robot is used in printing cDNA microarray
Large number of slides are placed on and secured to a platter
DNA samples are placed in microliter plates on the stand
Robot positions a cluster of specialized printing tips into
adjacent wells of DNA source plates, filling the reservoir slot
of each tip with approximately 1µ litre of DNA solution

Microarray Experimental Set Up

Printing of cDNA array
The cDNAs are printed on Poly-L-lysine coated glass
microscope slides
The arraying robot is used in printing cDNA microarray
Large number of slides are placed on and secured to a platter
DNA samples are placed in microliter plates on the stand
Robot positions a cluster of specialized printing tips into
adjacent wells of DNA source plates, filling the reservoir slot
of each tip with approximately 1µ litre of DNA solution
Tips are then lightly tapped at identical positions on each
slide leaving a small drop of DNA solution on the
poly-L-lysine coated slide

Microarray Experimental Set Up

After depositing DNA, the tips are washed and dried and the
process is repeated for the next DNA sample with a new spot,
at a small distance from the previous one to produce a high
density grid

Microarray Experimental Set Up

After depositing DNA, the tips are washed and dried and the
process is repeated for the next DNA sample with a new spot,
at a small distance from the previous one to produce a high
density grid
The speed of the robot is approximately 120 spots on 110
slides per hour

Microarray Experimental Set Up

After depositing DNA, the tips are washed and dried and the
process is repeated for the next DNA sample with a new spot,
at a small distance from the previous one to produce a high
density grid
The speed of the robot is approximately 120 spots on 110
slides per hour
Printing tips are the most critical components as they are the
only parts of the robot that touch the slide

Microarray Experimental Set Up

After depositing DNA, the tips are washed and dried and the
process is repeated for the next DNA sample with a new spot,
at a small distance from the previous one to produce a high
density grid
The speed of the robot is approximately 120 spots on 110
slides per hour
Printing tips are the most critical components as they are the
only parts of the robot that touch the slide
Tips operate on the same principle as a fountain pen; liquid is
drawn by capillary action and deposited when the tip makes
contact with the slide

Microarray Experimental Set Up

After depositing DNA, the tips are washed and dried and the
process is repeated for the next DNA sample with a new spot,
at a small distance from the previous one to produce a high
density grid
The speed of the robot is approximately 120 spots on 110
slides per hour
Printing tips are the most critical components as they are the
only parts of the robot that touch the slide
Tips operate on the same principle as a fountain pen; liquid is
drawn by capillary action and deposited when the tip makes
contact with the slide
cDNA in the spots is bonded to the glass to keep it from
washing off during the hybridization reaction and subsequent
wash

Microarray Experimental Set Up

Microarray Experimental Set Up
Many factors influence the size of the spot

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible
Most common problem is that the print tip gets clogged, fiber
or dust particles on the tip lead to spots of irregular shape

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible
Most common problem is that the print tip gets clogged, fiber
or dust particles on the tip lead to spots of irregular shape
Clean the tips

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible
Most common problem is that the print tip gets clogged, fiber
or dust particles on the tip lead to spots of irregular shape
Clean the tips
Tips become dull after some time and spots become large and
start overlapping each other

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible
Most common problem is that the print tip gets clogged, fiber
or dust particles on the tip lead to spots of irregular shape
Clean the tips
Tips become dull after some time and spots become large and
start overlapping each other
Change the tip or sharpen it

Microarray Experimental Set Up

Many factors influence the size of the spot
Sharper tips produce smaller spots, so the tips should be as
sharp as possible
Most common problem is that the print tip gets clogged, fiber
or dust particles on the tip lead to spots of irregular shape
Clean the tips
Tips become dull after some time and spots become large and
start overlapping each other
Change the tip or sharpen it
After printing, the area containing spots is marked with an
etching device

Microarray Experimental Set Up

(iii) Post processing of slides

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot
To distribute DNA more evenly, the spots are rehydrated and
snap dried

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot
To distribute DNA more evenly, the spots are rehydrated and
snap dried
Delicate process and insufficient rehydration can produce
irregular spot sizes while excessive rehydration can cause the
spots to fuse.

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot
To distribute DNA more evenly, the spots are rehydrated and
snap dried
Delicate process and insufficient rehydration can produce
irregular spot sizes while excessive rehydration can cause the
spots to fuse.
Blocking: Most important step of post processing

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot
To distribute DNA more evenly, the spots are rehydrated and
snap dried
Delicate process and insufficient rehydration can produce
irregular spot sizes while excessive rehydration can cause the
spots to fuse.
Blocking: Most important step of post processing
Remaining free reactive groups on the slide surface are
modified to minimize their ability to bind to labeled target
DNA

Microarray Experimental Set Up

(iii) Post processing of slides
Rehydration: Spotting process does not in general leave DNA
evenly distributed throughout the spot
To distribute DNA more evenly, the spots are rehydrated and
snap dried
Delicate process and insufficient rehydration can produce
irregular spot sizes while excessive rehydration can cause the
spots to fuse.
Blocking: Most important step of post processing
Remaining free reactive groups on the slide surface are
modified to minimize their ability to bind to labeled target
DNA
If these groups are not blocked, the labeled DNA target will
bind to the surface of the slide around the spot and produce
excessively high background
Microarray Experimental Set Up
(iv) Preparation of target: Isolate mRNA from the samples
under study and purify it. mRNA degrades very quickly, hence
sometimes it is reverse-transcribed into more stable cDNA.

Microarray Experimental Set Up

(iv) Preparation of target: Isolate mRNA from the samples
under study and purify it. mRNA degrades very quickly, hence
sometimes it is reverse-transcribed into more stable cDNA.
(v) Hybridization: Target sample is spinned into the micro
centrifuge and pipetted on the centre of the array without
forming any bubbles and allowed to diffuse all over the slide

Microarray Experimental Set Up

(iv) Preparation of target: Isolate mRNA from the samples
under study and purify it. mRNA degrades very quickly, hence
sometimes it is reverse-transcribed into more stable cDNA.
(v) Hybridization: Target sample is spinned into the micro
centrifuge and pipetted on the centre of the array without
forming any bubbles and allowed to diffuse all over the slide
A cover slip is placed on it and then the slide is kept in a
hybridization chamber for some period which is enough
incubation time for the hybridization reaction to take place

Microarray Experimental Set Up

(iv) Preparation of target: Isolate mRNA from the samples
under study and purify it. mRNA degrades very quickly, hence
sometimes it is reverse-transcribed into more stable cDNA.
(v) Hybridization: Target sample is spinned into the micro
centrifuge and pipetted on the centre of the array without
forming any bubbles and allowed to diffuse all over the slide
A cover slip is placed on it and then the slide is kept in a
hybridization chamber for some period which is enough
incubation time for the hybridization reaction to take place
In this process, a single stranded DNA molecule will bind with
highest affinity to another single stranded DNA molecule with
a precisely matching sequence and with significantly lower
affinity to one with an imperfect match

Microarray Experimental Set Up

(iv) Preparation of target: Isolate mRNA from the samples
under study and purify it. mRNA degrades very quickly, hence
sometimes it is reverse-transcribed into more stable cDNA.
(v) Hybridization: Target sample is spinned into the micro
centrifuge and pipetted on the centre of the array without
forming any bubbles and allowed to diffuse all over the slide
A cover slip is placed on it and then the slide is kept in a
hybridization chamber for some period which is enough
incubation time for the hybridization reaction to take place
In this process, a single stranded DNA molecule will bind with
highest affinity to another single stranded DNA molecule with
a precisely matching sequence and with significantly lower
affinity to one with an imperfect match
If the target contains cDNA whose sequence is complementary
to the DNA on a given spot on the microarray, that cDNA
will hybridize to the spot
Microarray Experimental Set Up
After hybridization, the microarray is thoroughly but carefully
washed to eliminate any excess labeled sample

Microarray Experimental Set Up

After hybridization, the microarray is thoroughly but carefully
washed to eliminate any excess labeled sample
Finally, it is dried using a centrifuge or by blowing with clean
compressed air

Microarray Experimental Set Up

After hybridization, the microarray is thoroughly but carefully
washed to eliminate any excess labeled sample
Finally, it is dried using a centrifuge or by blowing with clean
compressed air
To detect which cDNAs are bound to the microarray, the
target sample is labeled with a reporter molecule that flags
their presence

Microarray Experimental Set Up

After hybridization, the microarray is thoroughly but carefully
washed to eliminate any excess labeled sample
Finally, it is dried using a centrifuge or by blowing with clean
compressed air
To detect which cDNAs are bound to the microarray, the
target sample is labeled with a reporter molecule that flags
their presence
The reporters currently used in microarrays are fluorescent
dyes, called fluors or fluorophores

Microarray Experimental Set Up

After hybridization, the microarray is thoroughly but carefully
washed to eliminate any excess labeled sample
Finally, it is dried using a centrifuge or by blowing with clean
compressed air
To detect which cDNAs are bound to the microarray, the
target sample is labeled with a reporter molecule that flags
their presence
The reporters currently used in microarrays are fluorescent
dyes, called fluors or fluorophores
These are chemicals that fluoresce when exposed to a specific
wavelength of light

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target
Such an experiment is known as single channel microarray
experiment

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target
Such an experiment is known as single channel microarray
experiment
Sometimes two types of target mRNA samples are
simultaneously hybridized on the array. In such situation two
types of reporter molecules are added to two targets

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target
Such an experiment is known as single channel microarray
experiment
Sometimes two types of target mRNA samples are
simultaneously hybridized on the array. In such situation two
types of reporter molecules are added to two targets
Commonly used reporters are fluorescent dyes like Cy3 and
Cy5. These can be separated spectrally and they brightly
fluoresce

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target
Such an experiment is known as single channel microarray
experiment
Sometimes two types of target mRNA samples are
simultaneously hybridized on the array. In such situation two
types of reporter molecules are added to two targets
Commonly used reporters are fluorescent dyes like Cy3 and
Cy5. These can be separated spectrally and they brightly
fluoresce
Cy3 shows the green color and Cy5 shows red color when
excited by laser light at specific wavelength

Microarray Experimental Set Up

Sometimes a single type of target mRNA sample is hybridized
on the array. In such situation only one type of reporter
molecules are added to the target
Such an experiment is known as single channel microarray
experiment
Sometimes two types of target mRNA samples are
simultaneously hybridized on the array. In such situation two
types of reporter molecules are added to two targets
Commonly used reporters are fluorescent dyes like Cy3 and
Cy5. These can be separated spectrally and they brightly
fluoresce
Cy3 shows the green color and Cy5 shows red color when
excited by laser light at specific wavelength
This process simplifies the image acquisition process
Microarray Experimental Set Up
When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment

Microarray Experimental Set Up

When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment
It is essential to distinguish between these two types of arrays
as some methods for further analysis differ as the type differs.
Each type of arrays has its pros and cons.
(vi) Slide imaging: In this step, the microarray is scanned to
detect and measure the fluorescent signal emitted at each
spot and thus to determine the amount of labeled sample
bound to each spot

Microarray Experimental Set Up

When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment
It is essential to distinguish between these two types of arrays
as some methods for further analysis differ as the type differs.
Each type of arrays has its pros and cons.
(vi) Slide imaging: In this step, the microarray is scanned to
detect and measure the fluorescent signal emitted at each
spot and thus to determine the amount of labeled sample
bound to each spot
Device used is laser scanning confocal microscope (scanner)

Microarray Experimental Set Up

When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment
It is essential to distinguish between these two types of arrays
as some methods for further analysis differ as the type differs.
Each type of arrays has its pros and cons.
(vi) Slide imaging: In this step, the microarray is scanned to
detect and measure the fluorescent signal emitted at each
spot and thus to determine the amount of labeled sample
bound to each spot
Device used is laser scanning confocal microscope (scanner)
Scanner has a laser producing light with wavelength
appropriate for excitation spectra of dyes being used

Microarray Experimental Set Up

When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment
It is essential to distinguish between these two types of arrays
as some methods for further analysis differ as the type differs.
Each type of arrays has its pros and cons.
(vi) Slide imaging: In this step, the microarray is scanned to
detect and measure the fluorescent signal emitted at each
spot and thus to determine the amount of labeled sample
bound to each spot
Device used is laser scanning confocal microscope (scanner)
Scanner has a laser producing light with wavelength
appropriate for excitation spectra of dyes being used
For cy3, it is green light with wavelength around 540nm

Microarray Experimental Set Up

When two types of target mRNA samples are simultaneously
hybridized on the array, the experiment is known as two
channel microarray experiment
It is essential to distinguish between these two types of arrays
as some methods for further analysis differ as the type differs.
Each type of arrays has its pros and cons.
(vi) Slide imaging: In this step, the microarray is scanned to
detect and measure the fluorescent signal emitted at each
spot and thus to determine the amount of labeled sample
bound to each spot
Device used is laser scanning confocal microscope (scanner)
Scanner has a laser producing light with wavelength
appropriate for excitation spectra of dyes being used
For cy3, it is green light with wavelength around 540nm
for cy5, it is red light with wavelength around 650nm
Microarray Experimental Set Up
Light passes through a standard microscope objective and
illuminates a single point on the slide

Microarray Experimental Set Up

Light passes through a standard microscope objective and
illuminates a single point on the slide
Emitted light gathered by the objective passes through a
series of filters, collimating lens and pinhole and is quantified
in a photomultiplier tube

Microarray Experimental Set Up

Light passes through a standard microscope objective and
illuminates a single point on the slide
Emitted light gathered by the objective passes through a
series of filters, collimating lens and pinhole and is quantified
in a photomultiplier tube
Slide is rapidly scanned over the laser beam and an image of
the array is obtained
For a single channel array, the array is scanned only once

Microarray Experimental Set Up

Light passes through a standard microscope objective and
illuminates a single point on the slide
Emitted light gathered by the objective passes through a
series of filters, collimating lens and pinhole and is quantified
in a photomultiplier tube
Slide is rapidly scanned over the laser beam and an image of
the array is obtained
For a single channel array, the array is scanned only once
In this image, the intensity of each spot is proportional to the
amount of mRNA from the sample, matching with cDNA
sequence of given spot

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye
Subsequently, the array is again illuminated with laser light
frequency that excites the other dye

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye
Subsequently, the array is again illuminated with laser light
frequency that excites the other dye
Intensity of each spot in the second image is proportional to
amount of matching mRNA present in the second sample

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye
Subsequently, the array is again illuminated with laser light
frequency that excites the other dye
Intensity of each spot in the second image is proportional to
amount of matching mRNA present in the second sample
For visualization purpose, most of the available software
creates a composite image by overlapping the two images
corresponding to two channels

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye
Subsequently, the array is again illuminated with laser light
frequency that excites the other dye
Intensity of each spot in the second image is proportional to
amount of matching mRNA present in the second sample
For visualization purpose, most of the available software
creates a composite image by overlapping the two images
corresponding to two channels
Gene expressed in a sample labeled with red dye and not
expressed in the other sample will produce a red spot and
vice-versa

Microarray Experimental Set Up

In a two-channel experiment, scanning is done in two phases
Array is illuminated with laser light that excites one of two
fluorescent dyes and an image is captured for the wavelength
of that dye
Subsequently, the array is again illuminated with laser light
frequency that excites the other dye
Intensity of each spot in the second image is proportional to
amount of matching mRNA present in the second sample
For visualization purpose, most of the available software
creates a composite image by overlapping the two images
corresponding to two channels
Gene expressed in a sample labeled with red dye and not
expressed in the other sample will produce a red spot and
vice-versa
Gene expressed in both samples will produce equal amount of
red and green intensities and spot is given yellow psudocolor
Microarray Experimental Set Up
Microarray Experimental Set Up
For spotted cDNA or spotted oligonucleotide arrays, a test
sample and a reference sample labeled with two different
fluorescent dyes are commonly simultaneously hybridized on
the same array

Microarray Experimental Set Up

For spotted cDNA or spotted oligonucleotide arrays, a test
sample and a reference sample labeled with two different
fluorescent dyes are commonly simultaneously hybridized on
the same array
Spots with more bound sample will have more reporters and
will therefore fluoresce more intensely, higher the intensity of a
color, higher is the gene expression

Microarray Experimental Set Up

For spotted cDNA or spotted oligonucleotide arrays, a test
sample and a reference sample labeled with two different
fluorescent dyes are commonly simultaneously hybridized on
the same array
Spots with more bound sample will have more reporters and
will therefore fluoresce more intensely, higher the intensity of a
color, higher is the gene expression
The experimental goal is to measure the relative amount of
fluorescence from each fluor used

Microarray Experimental Set Up

For spotted cDNA or spotted oligonucleotide arrays, a test
sample and a reference sample labeled with two different
fluorescent dyes are commonly simultaneously hybridized on
the same array
Spots with more bound sample will have more reporters and
will therefore fluoresce more intensely, higher the intensity of a
color, higher is the gene expression
The experimental goal is to measure the relative amount of
fluorescence from each fluor used
There are many ways to achieve this goal, the most obvious
being segmenting the image into a very large number of pixels
corresponding to each spot and then determining the
fluorescence intensity for each pixel for each dye and then
taking average intensity as the intensity of the spot

Microarray Experimental Set Up

For spotted cDNA or spotted oligonucleotide arrays, a test
sample and a reference sample labeled with two different
fluorescent dyes are commonly simultaneously hybridized on
the same array
Spots with more bound sample will have more reporters and
will therefore fluoresce more intensely, higher the intensity of a
color, higher is the gene expression
The experimental goal is to measure the relative amount of
fluorescence from each fluor used
There are many ways to achieve this goal, the most obvious
being segmenting the image into a very large number of pixels
corresponding to each spot and then determining the
fluorescence intensity for each pixel for each dye and then
taking average intensity as the intensity of the spot
The relative amount of hybridized DNA in the two samples is
just the ratio of these fluorescent intensities.
Microarray Experimental Set Up