CONCEPTS IN MICROARRAYS

Gaurav Shrivastava

AMITY INSTITUTE OF BIOTECHNOLOGY

 Structural Hierarchy: from cells to DNA

All living organisms consist of cells. Plants / Humans have

trillions of cells, whereas Yeast is a single celled organism.

Cells are of different types (trichomes, oil glands, hairs,

blood, skin, nerve) and each cell contains a complete copy of
the genome (the program for making the organism), encoded
in DNA.
 Structural Hierarchy: from cells to DNA

DNA molecules are long double-stranded chains; 4 types of

bases are attached to the backbone: adenine (A), guanine (G),
cytosine (C), and thymine (T). A pairs with T, C with G.
A gene is a segment of DNA that specifies how to make a
protein. Human DNA has about 30-35,000 genes; Rice - about
50-60,000
Exons are coding DNA (translated into a protein), exons can be
thought of program data and Introns are non-coding DNA, which
provide structural integrity and regulatory (control) function .
 Cell Nucleus

Chromosome

Protein Gene (mRNA), Gene (DNA)

single strand
Why Microarray ?
 Gene Expression
Cells are different because of differential gene expression.
Gene is expressed by transcribing DNA into single-stranded
mRNA and mRNA is later translated into a protein.
mRNA expression represents dynamic aspects of cell
Microarrays measure the level of mRNA expression, mRNA
is isolated and labeled with fluorescent protein, hybridized to
the target. The level of hybridization corresponds to light
emission, which is measured with a laser and produce a digital
analog.
 Measuring Gene Expression

Gene is expressed
by transcribing DNA
into single-stranded
mRNA and
mRNA is later
translated into a
protein.

Idea: measure the amount of mRNA to see which

genes are being expressed in (used by) the cell.
What is Microarray ?
 Microarrays: Principle
DNA sequences representing all the genes in an organism can be
placed on miniature solid supports and used as hybridization substrates
to quantitate the expression of all the genes represented in a complex
mRNA sample. The key to this method of measuring gene expression
is hybridization. Hybridization is a process of forming a double-
stranded DNA molecule between a single-stranded DNA probe and a
single-stranded target DNA. Microarray is an extensive form of
Southern hybridization technique in which thousands or tens of
thousands of genes can be studied in parallel. There are five basic
aspects of microarrays.
• coupling biomolecules to a platform
• preparing samples for detection
• hybridization
• scanning
• analyzing the data
 Microarray Technique
 The goal of most microarray experiments is to
survey patterns of gene expression by assaying the
expression levels of thousands to tens of thousands
of genes in a single assay.

 Typically RNA is first isolated from different tissues,

developmental stages, disease states or samples
subjected to appropriate treatments.

 The RNA is then labeled with flourescent dyes and

hybridized to the arrays using an experimental
strategy that allows expression to be assayed and
compared between appropriate sample pairs.
Microarray Technique

 Common strategies include the use of single array

with distinguishable fluorescent dye labels for the
individual RNAs.
 The arrays are scanned after hybridization and
typically TIFF (tagged information file format)
images are generated for each pair of samples to
be compared.
 These images are then analyzed to identify the
arrayed spots and to measure the relative
fluorescence intensities for each element.
Microarray :Different Types
There are different types of microarrays (called
platforms), but all have a high density and number of
biomolecules fixed onto a well-defined surface.
Oligonucleotide arrays
Individual nucleotides are loaded one by one onto the
chip, where they attach to previously loaded nucleotides
to form probe sequences.
cDNA array
cDNA probes are fixed to the chip using robots. cDNA
arrays do not require a full genome sequence and they
can be generated relatively easily from pre-sequenced or
anonymous cDNA clones. They can be made containing
only a subset of genes of particular interest .
 Experimental strategy
•Microscopic coated slides are arrayed with many hundreds or thousands of
DNA samples (PCR products), referred to as targets. PCR products generally
contains genes of some known function.
• For gene expression profiling mRNA is prepared from two different samples,
which may represent two different stages or a diseased and normal tissue.
•The mRNA isolated from the cell should reflect the current state of expression
of each gene within the tissue.
•mRNA is converted to cDNA via first strand labelling reaction.. The labeled
probes then hybridized to the targets.
•Each mRNA that has been expressed will hybridized to its respective target
and therefore the signal generated will be directly related to the expression level
in the sample.
•The differentially expressed genes will be identified based on their signal
intensity.
 Biological question
Differentially expressed genes
Sample class prediction etc.

Experimental design

Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg), (Gfg, Gbg)
Normalization
R, G
Estimation Testing Clustering Discrimination

Biological verification
and interpretation
Microarray Spotter
•Most of the Microarray Spotter follow a consistent format with
respect to layout.A robotic arm controls a print head , capable of
moving in x, y and z axes.The print head holds up to 48 dispensing
units.
•Material to be spotted is organized in 96/ 384 well print plates, held
in a hotel,which allows them to be taken out, used, and replaced one
by one.
•Higher viscosity buffers are used for efficient printing. They also
determine spot size by determining how much a droplet will spread.
DNA microarray robot enclosed in a temperature and humidity controlled environment. The temperature and humidity must
be closely regulated or the spot sizes will vary or the printing pins will dry out too quickly.
 The print head of spotter with 48 pins. Each pin has a small capillary
like slit in it which draws up the DNA solution by capillary action.
Spotting/Arraying/Printing
DNA samples are printed on glass microscope slides, because
glass is flat, rigid,non-absorbent and has low fluorescence.Nucleic
acid binds efficiently with chemical coated glass slides. The most
common surface coatings are aldehyde, amino or poly-L-lysine
groups.

Aldehyde
If the coating is of Aldehyde (H-C=O) groups, then a Schiff base
(H-C=N-DNA) is formed, therefore slides are preprocessed with
sodium borohydride. which converts unreacted aldehyde groups to
non-reactive primary alcohols (H2-C=OH).
Spotting contd…/
Amine
Aminated slides are treated with aminosilane, which yields a
positively charged coating (NH3+). This positive charge will bind to
unmodified DNA, which carries a negative charge due to the
presence of phosphate groups.Binding occurs along with the DNA
backbone , resulting in highly efficient ionic bond formation.
Poly -L-Lysine
Poly -L-Lysine creates a positively charged ,via a charged amino
group, to which unmodified DNA can bind similar to aminated
slides. Natural electrostatic binding (H3N+/-DNA) is supplemented
by covalent attachment (H-N-DNA where N is also bound to the
arrays) induced by heat or UV light.
cDNA microarrays

cDNA clones
Control Spots
Negative Control Spots
These spots are normally consist of DNA/RNA corresponding to
another organism, and are expected to fail to hybridize.
Hybridization to negative control spots normally indicates that
hybridization conditions are not stringent enough.
Positive Control Spots
These spots are normally genomic material from another organism
and their corresponding genes are also spiked into the target sample
at known concentrations before labeling. A failure to get signal from
such positive control spots indicates that either labeling was not
efficient or hybridization condition are too stringent.
Probe Preparation

The most commonly used probe labeling dyes are Cyanine fluorophores
Cy 3 and Cy 5. Both samples prepared simultaneously using different
colored dyes.

By using fluorophores with different emission wavelengths, it is possible

to hybridize both the control and experimental samples onto the same
array simultaneously.

It is important that approximately equal amounts of modified nucleotide

are incorporated into any samples being co-hybridized.
 Hybridization

Labeled probe is applied to a preprinted microarray surface and the

slide is incubated in a temperature controlled humid environment for
4-18 hours.
Then slide is washed with a series of buffers for removing any
nonspecific binding of target and probe.The stringency of the
hybridization and washes are critical to the optimization process.
The main factors influencing the efficiency of hybridization are
volume, probe concentration , time and processing.
The post hybridization process is important for removing loosely
bound probes .
Hybridization

 For hybridisation, an amount of each

labelling reaction is combined in one tube
with hybridisation solution, fragmentation
buffer, and control targets, then loaded onto
the microarray slide then, the microarray is
placed on top of the Hyb chamber (array
side up), and fixed in the Hyb chamber for
overnight (14-18 hours) incubation at 65 o C.
Washing Of Hybridized
Slides
 Following incubation the slides are
washed to remove non-specific bound
targets. Slides are then scanned in the
microarray scanner and the resulting
images processed using Feature
Extraction software.
Image Capture & Data
Extraction
Microarray scanners employ lasers to capture fluorescent images ,
they deliver a beam of light through a filter on microarray within a
small wavelength range enabling optimal excitation and emission
wavelengths for each dye.
The amplified light (PMT) generates an analog signal which is
converted into a digital signal.This signal translates to a number
representing the intensity of fluorescence from a given pixel.
A printed array hybridized with sample A & B
 Microarray Data
Analysis
 The hypothesis underlying microarray analysis is that the
measured intensities for each arrayed genes represent its relative
expression level.

 Biologically relevant patterns of expression are typically identified

by comparing measured expression levels between different
states on a gene by gene basis.

 But before the levels can be compared appropriately , a number

of transformations must be carried out on the data to eliminate
low quality measurements, to adjust the measured intensities to
facilitate comparisons, and to select genes that are significantly
differentially expressed between classes of samples.
Parameters of analysis
 Background intensity: Mean signal Intensity of corners
between spots.
 Normalization: Total intensity normalization by median.
 Detection limit: Mean + 3SD of all the –ve controls (no DNA).
 Short-listing of regulated genes: Genes with > 2 fold
difference in expression patterns were considered for further
analysis.

Image Quantitation:
Image Quant
Arrayvision
Data Analysis
Gene Maths
Gene Spring
 Gene Expression Data
On p genes for n slides: p is O(10,000), n is
O(10-100), but growing,
Slides
slide 1 slide 2 slide 3 slide 4 slide 5 …
1 0.46 0.30 0.80 1.51 0.90 ...
2 -0.10 0.49 0.24 0.06 0.46 ...
Genes 3 0.15 0.74 0.04 0.10 0.20 ...
4 -0.45 -1.03 -0.79 -0.56 -0.32 ...
5 -0.06 1.06 1.35 1.09 -1.09 ...

Gene expression level of gene 5 in slide 4

= Log2( Red intensity / Green intensity)

These values are conventionally displayed

on a red (>0) yellow (0) green (<0) scale.
 Quantification of expression

For each spot on the slide we calculate

Red intensity = Rfg - Rbg

(fg = foreground, bg = background) and

Green intensity = Gfg - Gbg

and combine them in the log (base 2) ratio

Log2( Red intensity / Green intensity)

 Normalization and
Transformation
The hypothesis underlying microarray analysis is that the measured intensities
or each arrayed genes represent its relative expression level.

Normalization is the first transformation applied to expression data which will

adjusts the individual hybridization intensities to balance them appropriately for
the meaningful biological comparisons.

Normalization is important to reduce differences because of unequal quantities

of starting RNA , differences in labelling or detection efficiencies between the
fluorescent dyes used, and systematic biases in the measured expression
levels.

Replicate Filtering
Replication is essential for identifying and reducing the variation in any
microarray experiment. Biological replicates use RNA independently derived
from
Replicate Filtering

Replication is essential for identifying and reducing the

variation in any microarray experiment.
Biological replicates use RNA independently derived
from distinct biological sources and provide both a
measure of the natural biological variability in the
system under study as well as any random variation
used in sample preparation.
Technical replicates include replicated elements within a
single array, multiple independent elements for a
particular gene within an array ( such as independent
cDNAs or oligos for a particular gene), or replicated
hybridizations for a particular sample.
Microarray Potential
Applications
Microarray technology has proving to be an invaluable tool for gene
expression analysis on a genomic scale. The ability to study changes
in the expression of thousands of genes simultaneously has made it
possible to attain a global view of a cell's transcriptional state and to
associate genes with predictive functions or specific physiological
conditions.

 new and better molecular diagnostics

 new molecular targets for therapy
 finding and refining biological pathways
 Identification
Identificationof
of
functions
functionsofof
Differential
Differentialgene
gene new
new genes
genes
expression
expression

Toxicity
Assessment Comparisons
Microarrays between two
different
developmental
stages
Medical
Medicaldignostics
dignostics
Drug
Targeting
Applications of Microarray
Technology
Gene chips facilitated a more
comprehensive and inclusive experimental approach in
which alterations in the state of entire genomes simultaneously
can be assayed during health and disease or in response
to a variety of stimuli. This ability to profile
changes in gene-expression levels under different conditions
makes microarrays the method of choice in many
fields. Some of these fields are disease fingerprinting, drug
targeting and evaluation, toxicity assessment, signal transduction
research and developmental cancer treatment and
finding and refining biological pathways. All of these potential uses increase
the demand for
microarrays in both academic and industrial settings.
Impact Of Gene Chips and
Microarrays
 One of the most important experimental approaches for
discovering the function of genes promises to be gene chips
and microarrays.

 After analysing a complex mRNA population extensive

databases of quantitative information has been generated
about the degree to which each gene responds to pathogens,
pests, drought, cold, salt, photoperiod and other environmental
variations. and also about the response of genes during
developmental processes such as germination and flowering.

 Information related with the response of genes against the

phytohormones, growth regulators, herbicides, and elicitors.
Impact Of Gene Chips and
Microarrays
 These databases of gene expression information will
provide insights into the pathways of genes that control
complex responses.

 If it is found that variation in the expression of certain

pathways or processes is associated with enhanced yield or
quality in one species , this may provide testable targets for
rational improvement in other species.

 This approach will be a first step towards understanding the

ecology of the genome in which the genome is viewed as a
whole and relationships of gene products to each other will
be considered from atleast one perspective (relative level of
expression)
 Impact of Gene Chips and
Microarrays
 Work with plant microarrays is just beginning but there is every reason to
believe that this approach will soon be a standard component of the
repertoire of plant biologists.

 One of the most important advances in plant improvement was the

discovery of hybrid vigor and the exploitation of this phenomenon in the
modern breeding programs.

 Comparisons can be made between whole genome microarrays of inbred

parental lines with the hybrids. Hybrids may exhibit significant differences in
the expression of clusters of functionally related genes and that different
hybrids will have different patterns of expression.
Impact Of Gene Chips and
Microarrays
 The accumulation of DNA microarray or gene chip data from
many different experiments will create a potentially very
powerful opportunity to assign functional information to genes
of otherwise unknown function.

 The conceptual basis of the approach is that genes that

contribute to the same biological process will exhibit similar
patterns of expression.

 Thus by clustering of genes based on the similarity of their

relative levels of expression in response to diverse stimuli or
development or environmental conditions, it should be
possible to assign hypothetical functions to many genes
based on the known function of other genes in the cluster
Plant Microarrays

 Arabidopsis is the first plant genome

which was sequenced and used as a
model plant.

 Rice is the model system of cereal plants

with the smallest genome that is about
466mb in size containing 50-60 thousand
genes.
Rational Plant
Improvement
Knowledge of the function of all plant genes, in
conjunction with future development of tools for
modifying and interrogating genomes, will lead
to development of a robust genetic engineering
discipline in which rational changes can be
designed.
Analysis and Results
AN IMAGE SHOWING DIFFERENTIAL
EXPRESSION OF GENES

 Genechip: Arabidopsis 22 k
 Sample : RNA sample from Mentha arvensis

Equal Up- Regulation Down- regulation

expression
Spot Finding of the Four Corners of the Array
Spatial Distribution of Significantly Up-Regulated and
Down-Regulated Features
Suggested Readings
•Options available-from start to finish-for obtaining data from
DNA microarrays II. Nat Genet. 2002 Dec;32 Suppl 2:481-9. by
Holloway AJ, Van Laar RK, Tothill RW, Bowtell DD.

•Quantitative monitoring of gene expression patterns with

complementary DNA microarray. Science 270:467-470
Shena et al.
Thank you