MICROARRAY

WHAT IS A MICROARRAY ?

A tool for analyzing gene expression which is

made up of a small membrane or glass or
plastic or silicon biochip containing samples of
thousands of oligonucleotides arranged in a
regular pattern
PRINCIPLE:

The core principle behind Microarray is

hybridization of a nucleic acid sample to a very
large set of oligonucleotide probes, which are
attached to a solid support to determine
sequence or to detect variations in a gene
expression.
TYPES OF MICROARRAYS
o DNA microarray
o Protein microarray
o Glyco chip
o Tissue chip
STEPS INVOLVED:
o Preparation of sample
o Construction of array
o Hybridization
o Detection of result
o Analysis of result
o Submitting the results to microarray database
DNA MICROARRAY
DNA microarray is a multiplex technology used
in molecular biology and in medicine
It consists of an arrayed series of thousands of
microscopic spots of DNA oligonucleotides, called
features/cells, each containing picomoles of a
specific DNA sequence
This can be a short section of a gene or other
DNA element that are used as probes to
hybridize a cDNA or RNA sample (target) under
high-stringency conditions
 Probe-target hybridization is usually detected
and quantified by detection of fluorophore, silver,
or chemiluminescence-labeled targets to
determine relative abundance of nucleic acid
sequences in the target.
PREPARATION OF SAMPLE
mRNA has been extracted from the cells or
tissues under study, it is converted into DNA by
the use of the reverse transcriptase enzyme

During this reaction, the DNA is labelled by the

incorporation of fluorescent or radioactive
nucleotides into the DNA

The most common dyes in use are Cy3(green)

and Cy5(red)

This labelled DNA is then hybridised to the

microarray slide
CONSTRUCTION OF MICRROARRAY
Probe is a oligonucleotide ~25 bases long

These probes are printed in a microscopic slide by

robotically in the specified position
There are three main methods for constructing
the arrays and they are:
Spotting of DNA fragments onto the slide
Microspotting

Ink jetting

Piezoelectric printing

Photolithography
SPOTTING OF DNA FRAGMENTS DIRECTLY
ONTO THE SLIDE

At neutral pH, DNA molecules are charged

negatively, and the pattern of charges suggests
that phosphates in the DNA backbone would be
expected to bind strongly to a positively charged
surface, leaving the bases facing towards the
solution.
Glass slides can be silanized (positively charged)
by immersing them in a 2% solution of 3
aminopropyltriethoxysilane (APTES) in acetone
or this can also be carried out using
paminophenyltrimethoxysilane.
TECHNIQUES FOR SPOTTING DNA ONTO
THE SLIDE

Microspotting:
Printhead will carry the sample and it touches the
surface of glass slide and delivers sample in fixed
location, and moves to next place.

Ink jetting:
Printhead will carry the sample and its tip will not
touch the surface of slide. Without touching it will
deliver the sample on predetermined position
HYBRIDIZATION

Hybridization
is the reaction that occurs
between the DNA (tagged with fluorescent
material) and the probes on the
microarray
DETECTION OF RESULT

In a fluorescent labelling technique, photons

absorbed by a dye molecule illuminated at a
specific wavelength are re-emitted (in part) as
radiation at a lower frequency that is measured
with a photodetector or scanned using a laser
scanning microscope, which illuminates each spot
of DNA and separately measures the
fluorescence.
 This produces data to determine the
hybridisation pattern which can be used to
identify the genes that are expressed differently
in the tissues or cells.

A multitude of fluorophore dyes with spectrally

unique signatures have been developed for high
sensitivity biological labelling studies.
SUBMITTING THE RESULTS TO
MICROARRAY DATABASE

MGED (Microarray Gene Expression

Database):
It a consortium which is has been
established to address some of the issue imposed
by microarray data sharing.
PROTEIN MICROARRAY
Protein microarray technology enables high
throughput analysis of protein functions, such as
interactions between proteins, catalysis, binding
to drugs and other biochemical reactions.

The speed, precision, affordability and efficiency

of microarray analysis offer a tremendous
experimental advantage over traditional,
analytical tools using columns, gels, filters and
microplates.
 Protein microarrays are used in the study of
nucleic acid-protein, protein-protein, ligand -
receptor, drug protein target, and enzyme-
substrate interactions.
PREPARATION OF MICROARRAY
Optically flat, 96well glass plates were functionalized
with amine groups by immersing them in a solution of
aminopropyltrimethoxysilane (APTMS).

Proteins were printed onto the activated substrates

robotically, resulting in their covalent attachment to
the surface.

Unbound proteins are washed away, the substrates

were incubated with a solution of casein in phosphate
buffered saline (PBS) to minimize the non-specific
adsorption of proteins in subsequent steps.
 Sandwich
immunoassay
Antigen antibody Protein protein
interaction interaction

Nucleic acid protein Enzyme substrate

Ligand-receptor
interaction interaction
interaction
GLYCO CHIP
Very important in analysis of biomedical
problem.
Carbohydrate is diverse structure, inside the
body associated with different types of proteins
with different types of interaction.
This interaction can be used for analysis of
different types of carbohydrate-protein
interaction.
On microchip proteins are arrayed and
carbohydrates labelled used as probe and
interaction between protein and carbohydrate are
analyzed.
TISSUE CHIP
Tissue microarrays (TMAs) are used to analyze
the expression of genes simultaneously in
multiple individual tissue samples on one slide.

TMAs are composed of small 0.6 - 3.0 mm cores of

tissue from donor tissue paraffin blocks which
are arrayed at a high density on a slide.

Tissue Microarrays have allowed tissue

traditional analysis of conventional histological
paraffin blocks to become miniaturized and high-
throughput.
 Histochemical and molecular detection
techniques can be used on the TMA slides, and
thus allow TMAs to be powerful tools to examine
gene expression profiling in disease states across
a variety of patients and disease conditions.
CONSTRUCTION OF A TMA

Manufacturing TMAs is a fourstep process

Sample collection
Preparation of recipient blocks
Construction of TMA blocks
Sectioning.
SAMPLE COLLECTION
Exactly define the TMA to be made. Include
normal tissues.
Collect all slides of these tissues from the
archive.
One pathologist must review all sections from all
candidate specimens to select the optimal slide.
Tissue areas suited for subsequent punching
should be marked.
Collect the tissue blocks that correspond to the
selected slides.
These blocks and their corresponding marked
slides must be matched and sorted in the order of
appearance on the TMA.
PREPARING RECIPIENT BLOCKS
Melt paraffin at 60C, filtrate and pour it into a
stainless steel mould. In contrast to normal
paraffin blocks, tissue microarray blocks are cut
at room temperature. Therefore, a special type of
paraffin (PeelAWay paraffin; Polysciences
Inc., PA, USA) is recommended with a melting
temperature between 53 and 55C.
Place a slotted plastic embedding cassette (as
used in every histology lab) on the top of the
warm paraffin.
Cool paraffin block down for 2 hours at room
temperature and for 2 additional hours at 4C.
Large recipient blocks (for example 304510
mm) are easier to handle than the smaller blocks.
TMA BLOCK CONSTRUCTION
The TMA manufacturing process consists of five
steps that are repeated for each sample placed on
the TMA:
Punching a hole into an empty (recipient) paraffin
block removing and discarding the wax cylinder from
the needle used for recipient block punching
Removing a cylindrical sample from a donor paraffin
block
Placing the cylindrical tissue sample in the pre-made
hole in the recipient block
Proceeding to the new coordinates for the next tissue
sample
TMA BLOCK SECTIONING
Place an adhesive tape on the TMA block in the
microtome immediately before cutting.
Cut a section (usually 5 m). The tissue slice is
now adhering to the tape.
Place the tissue slice on a special glued slide

Expose the slide (tissue on the bottom) to UV

light for 35 seconds (This leads to polymerization
of the glue on the slide and on the tape).
Dip the slide into TPC solution (Instrumedics) at
room temperature for 510 seconds.
Gently remove the tape from the glass slide
leaving the tissue on the slide.
Air dry slides at room temperature.
Thank you