Microarray

Dr. Swarnendu Pal

 Background
 History
 Introduction
 Types of microarrays
 DNA microarray
 Principle
 Types of DNA microarray
 Steps involved in DNA microarray
 Applications
 Advantages
 Limitations and disadvantages
 Future of microarrays
 Take home message
 Background
 A human organism has over 250 different cell types
(e.g., muscle, skin, bone, neuron), most of which
have identical genomes, yet they look different and
have different activities

 Less than 20% of the genes are expressed in a typical

cell type

 Apparently the differences in gene expression is what

makes the cells look and function differently
 History
• Once the human genome sequence was completed
in 2001, it paved the way for many experiments
and researches;

• one such area was identifying the regions of DNA

which control normal and disease states.

• Functional genomics is the study of gene function

through parallel expression measurements of a
genome.
 History
• Microarray  analyzes large amount of samples which
have either been recorded previously or from new
samples

• History: Microarray technology evolved from Southern

blotting, where fragmented DNA is attached to a
substrate and then probed with a known DNA
sequence

• Use of miniaturized microarrays for gene expression

profiling first reported in 1995, and a complete
eukaryotic genome (Saccharomyces cerevisiae) on a
microarray was published in 1997
 Microarray
• It is a 2D array on a solid
substrate (usually a glass
slide or silicon thin-film
cell) that assays large
amounts of biological
material using high-
throughput screening,
miniaturized,
multiplexed and parallel
processing and detection
methods.
 Types
 DNA microarrays, such as cDNA microarrays,
oligonucleotide microarrays, BAC microarrays and SNP
microarrays
 MMChips, for surveillance of microRNA populations
 Protein microarrays
 Peptide microarrays, for detailed analyses or optimization
of protein–protein interactions
 Tissue microarrays
 Cellular microarrays(also called transfection microarrays)
 Chemical compound microarrays
 Antibody microarrays
 DNA microarray
• A DNA microarray (also commonly known as
genome chip, DNA chip, or gene array) is a
collection of microscopic DNA spots, commonly
representing single genes, arrayed on a solid
surface by covalent attachment to a chemical
matrix.

• Each spot of DNA, called a probe, represents a

single gene.
 Principle
• Hybridization : The property of complementary
nucleic acid sequences is to specifically pair
with each other by forming hydrogen bonds
between complementary nucleotide base pairs.

• The principle of DNA microarrays lies on the

hybridization between the nucleotide. Using this
technology the presence of one genomic or cDNA
sequence in 1,00,000 or more sequences can be
screened in a single hybridization.
 Types of DNA microarrays
• Based on different technologies and probe types,
and intended application it can be:

 Bacterial artificial chromosome arrays

 cDNA microarrays
 Oligonucleotide microarrays
 In-situ synthesized microarrays
 Bead microarrays
 Steps involved
In microarray

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
Instruments required
 Steps involved • Collecting tissue
In microarray

I. Collect tissue
II. Isolate RNA
III. Isolate mRNA
IV. Make labeled DNA copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating RNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating RNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating RNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating RNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating RNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating mRNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating mRNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved • Isolating mRNA
In microarray

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray • Isolating mRNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Make labeled DNA copy

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Make labeled DNA copy

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Make labeled DNA copy

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Make labeled DNA copy

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Apply DNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Apply DNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Apply DNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Apply DNA

I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Steps involved
In microarray Scan
microarray
I. Collect tissue
II. Isolate RNA
III.Isolate mRNA
IV. Make labeled DNA
copy
V. Apply DNA
VI. Scan microarray
VII. Analyze data
 Analyzing data
• GREEN represents Control DNA, where either DNA
or cDNA derived from normal tissue is hybridized to
the target DNA.
• RED represents Sample DNA, where either DNA or
cDNA is derived from diseased tissue hybridized to
the target DNA.
• YELLOW represents a combination of Control and
Sample DNA, where both hybridized equally to the
target DNA.
• BLACK represents areas where neither the Control
nor Sample DNA hybridized to the target DNA.
Analyzing data
Image Analysis & Data Visualization
 Some questions for the age of genomics

• How gene expression differs in different cell types?

• How gene expression differs in a normal and diseased (e.g.,

cancerous) cell?

• How gene expression changes when a cell is treated by a

drug?

• How gene expression changes when the organism develops

and cells are differentiating?
 Applications
 In cancer

 Tumor formation involves simultaneous changes in

hundreds of cells and variations in genes.

 Identification of single-nucleotide polymorphisms

(SNPs) and mutations, classification of tumors,
identification of target genes of tumor suppressors,
 Applications
 In cancer

 Identification of cancer biomarkers, identification

of genes associated with chemoresistance

 Early detection of precancerous lesions

 Identification of gene expression profiles or

“genomic fingerprints” will allow clinicians to
differentiate harmless lesions from precancerous
lesions or from very early cancer
 Applications
 Antibiotic treatment
 Gene expression profiling
 In different cells/tissues
 During the course of development
 Under different environmental or chemical stimuli
 In disease state versus healthy
 Molecular diagnosis
 Molecular classification of disease
 Drug development
 Identification of new targets
 Pharmacogenomics
 Individualized medicine
 Advantages
 Provides data for thousands of genes.

 One experiment instead of many.

 Fast and easy to obtain results.

 Huge step closer to discovering cures for diseases

and cancer.

 Different parts of DNA can be used to study gene

expresion
 Limitations of DNA microarrays
 First, arrays provide an indirect measure of relative
concentration.
However, due to the kinetics of hybridization, the
signal level at a given location on the array is not
linearly proportional to concentration of the species
hybridizing to the array.

 For complex mammalian genomes, it is often difficult

to design arrays in which multiple related DNA/RNA
sequences will not bind to the same probe on the
array.
 Disadvantages
 The biggest disadvantage of DNA chips is that they are
expensive to create.

 The production of too many results at a time requires

long time for analysis, which is quite complex in
nature.

 The DNA chips do not have very long shelf life, which
proves to be another major disadvantage of the
technology
 The Future of DNA arrays
 When the cost is similar, sequencing has many advantages
relative to microarrays.
Sequencing is a direct measurement of nucleic acids
present in solution. One need only count the number of a
given type of sequences present to determine it’s
abundance.

 Unlike DNA arrays, sequencing is not dependent on prior

knowledge of which nucleic acids may be present.
 The Future of DNA arrays
 Sequencing is also able to independently detect closely
related gene sequences, novel splice forms or RNA
editing that may be missed due to cross hybridization
on DNA microarrays

 As a result of these advantages and the decreasing cost

of sequencing, DNA arrays are being rapidly replaced by
sequencing for nearly every assay
 Take home message
 Microarrays are a powerful tool and holds much
promise for the analysis of diseases.

 Classifications of disease by DNA, RNA, or protein

profiles will greatly enhance our ability to diagnose,
prevent, monitor and treat our patients.

 Microarrays promise a more biologically based,

individualized treatment
Thank You