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DNA Microarray analysis

Principles of microarray analysis
The technical solutions that have been developed for performing microarray analysis, all
are miniaturized hybridization assays for studying thousands of nucleic acid fragments
simultaneously. All microarray systems share the following key components:

the array, which contains immobilized nucleic acid sequences, or ‘targets’

one or more labeled samples or ‘probes’, that are hybridized with the microarray
a detection system that quantitates the hybridization signal

Manufacturing of microarray slides

Introduction
Microarray analysis is performed on a glass slide. This enables the performance of
hybridization assays with uorescently labeled samples.

Microarray manufacture requires three distinct components:

production method
microarray slide
target genetic content

 Production methods
Oligo synthesis
Two parallel approaches have been developed for the production of microarray slides.
Nucleic acid targets can either be synthesized directly onto the microarray slide, or puri ed
targets can be deposited onto a solid surface that is capable of binding nucleic acids.

Oligo synthesis is done by attaching chemically modi ed linker groups onto the glass
surface.  These linker groups contain photochemically removable protective substances.
By masking di erent prede ned positions in di erent steps, we can synthesize di erent
nucleotides at various regions. Target synthesis proceeds in a step-wise fashion. In each
step, the unprotected areas are rst activated with light to remove the light-sensitive
protective groups.

This method produces arrays of small features that are anchored at their 3′ ends to the
array surface. Each feature is made up of oligonucleotides that all have the same
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nucleotide sequence. These arrays have a high density: an area of 1.6 cm can contain up to
4,00,000 features.

Deposition
Using common deposition methods, puri ed nucleic acids are attached to a modi ed glass
slide. Typically, small volumes of nucleic acid solution are transferred onto the glass slide.
Deposition methods are equally suitable for preparing microarrays containing
oligonucleotides, cDNA sequences, as well as genomic DNA.

The deposition involves the formation of covalent bonds between the oligo and the glass
surface. It binds the oligonucleotide onto the array. The manufacturing process should
meet several criteria. Variation in the quantity of targets deposited, the shape of spots, the
regularity of the array pattern, and the carryover of targets could all detrimentally a ect
the accuracy of microarray data.

Non-contact deposition
The printing heads do not touch the surface of the microarray. Piezoelectric printing and
syringe-solenoid methods are the two common variations of this method.

■ In piezoelectric printing, the target solution is drawn into a capillary that is in contact
with a piezoelectric crystal. Application of the voltage to the crystal results in a slight
conformational change, squeezing the capillary.

■ Syringe-solenoid deposition uses a syringe pump positive displacement method to

deposit nano liter volumes onto a slide. A syringe that provides the pressure source is
connected to a micro-solenoid valve.

Contact deposition
In contact deposition, solid, hollow, or split-open pen designs are used to transfer target
nucleic acid onto the slide surface. These pens are dipped into the target solution, a small
volume of which adheres to the pen. When the pen comes into contact with the slide
surface, a fraction of the nucleic acid solution on the pen is deposited onto the glass
surface.

Slide surface chemistries

Nucleic acids will not attach e ciently to an untreated glass slide. Di erent chemical
treatments have been developed to facilitate the attachment. These treatments have an
in uence on the density of the molecules that can be attached.Uniformity and thickness of
the surface coating are critical quality parameters. These in uences spot uniformity, DNA
binding, and noise to signal ratio.  Commonly used slide surface modi cations include the
introduction of aldehyde, amino, or poly-lysine groups onto the slide surface.

Common slide types

Aldehyde slides

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Amino-modi ed DNA can be attached to microarray slides that have been modi ed with
aldehyde groups. The amino group can be introduced into DNA in a PCR ampli cation
reaction using amino-modi ed oligonucleotides. The aliphatic amine on the amino-
modi ed DNA acts as a nucleophile. It attacks the carbon atom aldehydes, which is
covalently attached to the surface of the slide. An unstable intermediate is converted to a
Schi base through a dehydration reaction (-H2O), and the DNA is bound to the surface.
The unreacted aldehyde groups are reduced to non-reactive primary alcohols by
treatment with sodium borohydride (NaBH4).

Amine slides
Amine groups can be introduced onto microarray slides by treating cleaned glass with
aminosilane, such as 3-aminopropyltrimethoxysilane. Unmodi ed DNA can be attached to
amine-modi ed slides, via interactions between negatively charged phosphate groups on
the DNA and the positively charged slide surface. This interaction also denatures the DNA
which increases its binding a nity. UV treatment is used to further immobilize the DNA
onto the slide surface. Attachment via electrostatic interactions is suitable for binding DNA
fragments that are longer than 60–70 nucleotides. For attaching oligonucleotides to amine-
modi ed glass, chemical coupling methods must be used.

Poly-lysine slides                                                                
Treatment of the slide with poly-lysine creates a positively charged surface to which
unmodi ed DNA can bind via ionic interactions.

Re ective slides
A large proportion of the uorescent light emitted from the hybridized probe is scattered
in all directions when using regular glass arrays. The introduction of a re ective surface
below the spotting surface enables a signi cant amount of this scattered output to be
directed towards the detector, hence increasing the amount of signal detected by the
system. These re ective slides are constructed by adding a layer of aluminum above the
glass surface.

Target nucleic acids

The third critical component in microarray manufacturing is the target nucleic acid. A high
concentration of target molecules for deposition. The purity of target solutions is
important for both the e cient attachment of nucleic acids to the slide surface and the
availability of the immobilized targets for hybridization.  The targets are available for
hybridization only in single stranded denatured form. This can be achieved by spotting the
targets under denaturing conditions. Typically, targets are dissolved in high salt solutions
such as 3 × SSC, or in denaturing solvents such as DMSO.

Requirements of labeling methods

Retaining gene expression information

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Any labeling methods used in the microarray must cope with the inherent diversity of the
transcript sequences. It should contain all the information of the original transcript
population. An ideal labeling system is neither biased towards any nucleotide sequences,
nor does it label di erently transcripts of di erent sizes or sequences that are expressed
at di erent levels. In reality, existing labeling methods do not convert all information into a
labeled form. Enzymatic methods are limited to copying certain nucleic acid sequences,
whereas the instability of some transcripts is a general problem for all methods

Length of labeled fragments

Hybridization of nucleic acids and speci city for the complementary targets should be
retained for the extraction of accurate information about gene expression.  The length of
the labeled fragment is an important factor in determining these parameters. For optimal
hybridization, probes consisting of fragments of range 200-500 nucleotides sequence are
recommended. Longer fragments may not nd their targets as e ciently as shorter
fragments.

Yield of labeled probe

The amount of labeled probe prepared by the labeling method is important to the
sensitivity of microarray experiments. This is because the e ciency of the labeling process
is critical in determining the lowest amount of mRNA that can be used to generate
detectable signals from microarrays. The e ciency can make the di erence between being
able to hybridize one or several slides with one probe. Ideally, the labeling method should
transform each transcript into a labeled fragment, without any bias towards more highly
expressed sequences. If the labeling method results in net ampli cation of nucleic acid in
the labeling process, the ampli cation process should be linear, i.e. the original ratios of
expression levels within the sample should not be changed in the ampli cation process.

Optimum labeling density

If two or more uorescent molecules are in close proximity to each other, a signi cant
portion of the absorbed light energy can be spent on interactions between di erent
molecules and dissipated as heat. This will result in the reduction of the amount of
uorescence and it is no longer directly proportional to the number of the uorophore in
the sample. This phenomenon is called “quenching”, and it is an inherent property of
uorescent molecules. Each uorophore has slightly di erent quenching properties. In
practical terms, this means that for each uorophore there is an optimal labeling density,
or distance between attached labels, which will produce maximum uorescence from a
labeled nucleic acid fragment. Exceeding this optimum labeling density results in a
decreased uorescent signal. Thus optimal optimization density is required in all labeling
methods.

Measuring the amount of CyDye in the probe with

spectrophotometry
Follow the steps below to measure the amount of incorporated CyDye:
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Sample preparation
Dilute an aliquot of a puri ed probe with water. The required volume depends on the size
of available measuring cells. It is recommended to use the smallest cells possible. They
minimize cross-contamination from one sample to another. If 100 μl glass cells are used
cleaning thoroughly by rinsing with sterile water between samples.

Measurement
1. Measure the absorbance spectrum of the sample from 200 to 700 nm against a blank.
The absorption spectrum of CyDye contains two peaks, of which the second should be of
higher intensity. The rst peak indicates the presence of intermolecular interactions
between di erent dye molecules. If both peaks are of nearly similar intensity, this is a sign
of quenching. This is usually associated with over labeling of the sample.
2. In case of dual labeling, measuring absorbance at 550 nm for Cy3 and at 650 nm for
Cy5. The observed absorbance values depend on how much the sample was diluted
before measurement, how much RNA was used in the labeling, the labeling method used,
and the e ciency of labeling. Typically, values around 0.050 would be expected from rst-
strand cDNA labeling reactions in which 1 μg of mRNA is used as a template.

Recover the measured sample for future use. It may be necessary to concentrate the
sample before using for microarray hybridization. This can be performed by drying
down the sample in a vacuum concentrator or by letting sample that has been heated to
60 °C evaporate to dryness. It is important to protect the sample from light during all
handling.

Calculation
The amounts of Cy3 and Cy5 incorporated into probes can be calculated from their
respective extinction coe cients and using the following equations:

Extinction coe cients

150 000 M–1 cm–1 at 550 nm for Cy3 and

250 000 M–1 cm–1 at 650 nm for Cy5

Calculation equations

pmol Cy3 in puri ed sample = (A550 / 150 000) × dilution factor × (z μl) × (w cm) × 1012

where

A550 = absorbance at 550 nm

z μl =  volume of the sample after puri cation

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w cm = optical path in cuvette

Cy5 (p mol in sample) = (A650 / 250 000) × dilution factor × (z μl ) × (w cm) × 1012

where

A650 = absorbance at 650 nm

z μl =  volume of the sample after puri cation

w cm = optical path in cuvette

Determination of the amount of labeled nucleic acid

in the sample
UV spectrophotometry
Two methods can be used to determine the amount of labeled nucleic acid in the sample:
spectrophotometry and radioactive spiking. Nucleic acids absorb at 260 nm, and
absorption measurement at this wavelength can be used for quantitative purposes.
Distortions may happen due to impurities. Therefore absorption spectra from 200 to 400
nm should be measured, and only if the peak at 260 nm is clearly distinguishable from
absorption at near wavelengths, should estimation of nucleic acid amount be made

The following approximations can be used to calculate the amount of nucleic acid in the
probe:

1 A260 unit of double-stranded DNA corresponds to 50 μg/ml

1 A260 unit of single-stranded DNA corresponds to 37 μg/ml

1 A260 unit of single-stranded RNA corresponds to 40 μg/ml

Spiking with radioactive nucleotide

For more accurate quanti cation of labeled nucleic acid labeling can be spiked with a small
amount of a radioactive nucleotide. This enables the determination of the amount of
nucleic acid synthesized as well as the amount of sample recovered from puri cation. This
approach can be used with all labeling methods in which new nucleic acid is synthesized.

Calculation of labeling density

Labelling density can be de ned as the amount of CyDye incorporated into a known
amount of nucleic acid.

Labelling density = pmol CyDye in labeled sample

μg nucleic acid in labeled sample

1 μg of cDNA contains approximately 1 × 10-6 /330 = 3030 pmol of nucleotides.

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Analyzing CyDye labeled probes with PAGE

In order to analyze CyDye labeled probes using PAGE, follow the procedures detailed
below:

Preparation
1. PAGE gels suitable for analysis of labeled samples are prepared from standard 6% or 8%
(w/v) sequencing gel mix. Slab gel instruments can be used. These provide the added
bene t of thicker and deeper wells that simplify sample loading.
2. 0.1–1 μl of puri ed labeling reaction is enough for detection of CyDye labeled nucleic
acid by uorescence scanning. Add 2 μl of formamide and 8 μl of water to each sample. Do
not use loading/denaturation bu ers which contain dyes such as bromophenol blue or
xylene cyanol, as these will interfere with the detection.

Dilute 4 μl of uorescent markers with 4 μl of water and 2 μl of formamide. This sizer

contains a Cy5-labelled DNA marker ladder.

Denature
Denature all samples by boiling for 2 min at 95 °C. Snap cool on ice before loading on to
the gel.

Electrophoresis
1. Load samples to a gel that has been pre-electrophoresed for 15–30 min. Perform
electrophoresis according to the instructions provided with your equipment. Use 1× TBE as
the bu er.
2. A marker with 1 μl bromophenol blue is used.It also aids in the runtime of the
electrophoresis. Keep the bromophenol blue within the gel.

Remove and clean the gel plate. Do not let the gel plates to go dry.

Imaging
Scan the gel on an Imager. Detect Cy3 by excitation with 532 nm laser and using emission
lter 555 BP 20. Detect Cy5 by excitation with 633 nm laser and using emission lter 670
BP 30. Set PMT to 800 V, focal plane to +3 mm, and use normal sensitivity. The PMT values
may need to be adjusted to account for di erent amounts of sample in the gel.

Hybridization
The process of hybridization is typically performed in order to identify and quantitate
nucleic acids within a larger sample. It involves annealing a single-stranded nucleic acid to
a target complementary strand. Southern blotting is one well-established hybridization
method. The target nucleotide sequence is attached to a membrane and hybridized with a
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labeled probe. Fluorescence con rms the presence of target sequence and its intensity is
proportional to a number of a nucleotide sequence.

There are several critical factors to performing a successful microarray hybridization.

■ Pre-hybridization

■ Hybridization conditions

■ Hybridization bu er

■ Stringency washes

Pre-hybridization
Thi steps consist of incubating the DNA microarray without a probe. Preincubation is done
for the following reasons. Badly Adhered DNA sequence is washed away. Pre hybridization
ensures the availability of the nucleotide sequence for hybridization. Blocking of non-
speci c hybridization is done by this step.

Hybridization Conditions
Coverslip Method
Hybridization is done under a coverslip. 30 μl, of the bu er, is used. Dying out of
nucleotide around the edges is a limitation. But it can be avoided by maintaining humid
experimental conditions. Humidity more than 95 % should be maintained. The
hybridization time is 16-hours.

Hybridization bu er
A Hybridization bu er should contain the following components:

1. A bu ering component that acts to stabilize variations in pH.

2. A detergent that acts to lower the surface tension and allow the bu er to ow easily
under a coverslip

A compound that act as rate enhancers, volume excluders, or to speed up the

hybridization and lower the melting point(Tm).

Probe concentration
Probe concentration may vary depending on due to the samples used, the slide type and
information requirement. For each slide type manufacturers would recommend an
optimum concentration of probes.

Probe depletion and target saturation

To provide complete isolation and movement, complete coverage of the array by the
bu er solution is mandatory. Once covered, di usion plays an important role in the

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movement of these probes to the target nucleotides. A 20 bp oligo nucleotide di uses to a

distance of 3.6 mm during the 18 h period. A cDNA sequence hardly moves throughout the
hybridization process. A high concentration of target sequence will lead to a competing
inhibition. This phenomenon is known as target depletion. It limits the signal received from
the microarray.  For a high abundance gene, the amount of probe in solution starts to
approach the amount of target present, which can lead to target saturation. Target
saturation will be determined by factors such as the amount of target initially spotted on
the slide and the amount retained on that slide after pre-treatment, as well as the
percentage of that target available for hybridization and the e ciency of hybridization.
Together, the sensitivity of detection and target saturation determine the dynamic range
of the microarray experiment.

Pre-hybridization
1. Store spotted slides in a desiccator until use.
2. Read through the pretreatment and hybridization protocols thoroughly before use, as
bu ers often need preparing and preheating before use.

Pretreat the number of slides required for the experiment.

1. During the pre-treatment stage, prepare the probes. This will often involve drying down
equivalent amounts of the two probes of interest together and reconstituting them in the
manufacturer’s recommended bu er. Some protocols require heating the probe before
use; the probe prepared by reverse transcription will be single-stranded and therefore
should not require denaturing before use.

Hybridization
1. Once the probe is prepared, lay the spotted slide, DNA side up, on a clean surface.
Absorbent tissue that does not release any bers is a good choice of surface. This is
important, as most slides are glass, and dirt on the rear of the slide will a ect the result on
the front of the slide.
2. Using a pipette, transfer the required amount of hybridization bu er/probe mixture
onto the slide. Avoid touching the slide surface with the pipette tip. Try to deposit the
mixture along with the short side of the slide, away from the spotted area.

Take a clean coverslip and place it on the slide near the probe mixture, and allow surface
tension to speed the bu er along the coverslip. Then gently lower the cover slip,
avoiding trapping air bubbles underneath.

1. If air bubbles become trapped beneath a coverslip, do not move the coverslip to try and
remove them. Movement of the coverslip will result in damage to the targets themselves.
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Most small air bubbles will disperse once the slide is transferred to hybridization
temperature. Larger bubbles can be ‘encouraged’ to move by gently pressing on the
surface of the coverslip with a pipette tip.
2. The probe mixture is light sensitive, so once the coverslip is on, place the slide in the
humid chamber and incubate overnight in the dark.

Stringency washes

The purpose of the post-hybridization washes is to remove all unattached and loosely
bound probe molecules. This prevents false positive signals and removes all components
of the hybridization bu er, preventing background noise in the form of smearing and
speckles.  Once the slides have been washed, they should immediately be dried by
centrifugation or nitrogen steam to prevent smearing while drying. The slides should then
be stored in the dark in a desiccator and scanned as soon as possible. If once scanned, it is
found that the slides have a high background or low stringency, it is worth rewashing the
slide and re-scanning.

Image analysis
A scanned microarray image records the uorescent intensities of both the signal and
background DNA spots. The rst step in data analysis is to identify these spots.
Measurement the e intensities of both the spot and background uorescence. This
process is known as gridding. It can be done by many software. It gives an output the size
of the spot, the distance between two spots and all other measurable quantities.

Controls
As with all experiments, micro arrays should contain a series of controls to ensure that the
data obtained from the arrays is accurate.

Negative controls
Negative controls are spotted DNA sequences that should not hybridize with any labeled
probe. The negative controls used should ideally come from organisms that are only
distantly related to the organism being studied in the experiment. Under optimal analysis
conditions, negative control spots should not give any signal at all.

Poly-adenylated DNA and CotI DNA

When using oligo(dT) to prime rst-strand cDNA synthesis, it is possible that the oligo(dT)
will prime within the poly-A tail of the mRNA. If this occurs there will be a string of dT bases
within the probe. In order to prevent cross reactivity of the poly-dT sequences within the
probe with potential poly-dA sequences in the targets, a poly-dA oligo of 80 bases can be
included in the hybridization to block the poly-dT.

Positive controls
Labelled DNA
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DNA can be labeled with CyDye uors using polymerase chain reaction (PCR), or in the
case of oligos, during the synthesis of the oligos (many oligo manufacturers o er this
service). When the labeled DNA is spotted onto the array, the DNA will be uorescent and
serve as a useful positive control for verifying that the target DNA is binding e ectively to
the slide surface during the hybridization and washes.

Normalization
In order to compare ratio data from one microarray slide to another microarray slide, the
ratio data needs to be normalized to correct for experimental variation. The reason for this
is that from one slide to another there will be di erences between the relative Cy3 and Cy5
signals due to one or more of the following:

the amounts of mRNA used in the Cy3 and Cy5 labeling reaction
the e ciency of detection of the Cy3 and Cy5 by the detection system within the scanner
relative incorporation di erences of the Cy3 and Cy5 reverse transcriptases

Visualization and clustering

After microarray data is normalized to account for di erences in Cy3 and Cy5 signals, it
can subsequently be exported to any number of data visualization software for further
analysis. These software products can be used to mine the data for signi cant changes in
gene expression. The process of visualization can signi cantly enhance data analysis. It can
provide helpful features, such as data integration, customized query devices, and pattern
recognition. Clustering data points, or genes, that show similar responses on microarray
analysis can be used to identify genes that have similar gene expression patterns and
which possibly belong to the same pathway.

Search

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