DNA microarray technology allows researchers to analyze gene expression patterns across thousands of genes simultaneously. It involves affixing DNA probes to a solid surface in an orderly array and then measuring which genes are expressed by the level of hybridization with fluorescently labeled cDNA or cRNA from samples. The document discusses the history and principles of microarray techniques, including types such as cDNA and oligonucleotide microarrays. It also covers applications in genomics research and analysis of microarray data.

1. MICROARRAY TECHNIQUE
ARUN CHACKO
PhD SCHOLAR
PLANT BREEDING & GENETICS

2. An array
• Orderly arrangement of samples where
matching of known and unknown DNA
samples is done based on base pairing rules.
• ADNAmicroarray (DNAchip or biochip)is a
collection of microscopic DNAspots attached to a
solid surface.
• An array experiment makes use of common
assay systems such as microplates or standard
blotting membranes.

3. History of Microarray
• 1965 : Gillespie and Spiegelman described
methods for DNA blotting hybridization, in
which DNA immobilized on a membrane can
bind to complementary RNA or DNA strand
through specific hybridization.
• 1975: Southern blotting developed by E. M.
Southern

4. • Microarray technology evolved from Southern
blotting.
• The concept of microarrays was first proposed in
the late 1980s by Augenlicht and his colleagues.
• They spotted 4000 cDNA sequences on
nitrocellulose membrane and used radioactive
labeling to analyze differences in gene expression
patterns among different types of colon tumors in
various stages of malignancy.

5. • 1982: RNA was isolated from normal and
cancer tissue of mice, cDNA synthesized,
cloned on E. Coli and 378 colonies were
arrayed.
• 1991: Scientists at the California-based biotech
company Affymetrix produce the first DNA
chips.

6. • 1995: Quantitative monitoring of gene
expression patterns with a complementary
DNA microarray.
• 1995:microarrays for gene expression profiling
was done using complete eukaryotic genome
(Saccharomyces cerevisiae) on a microarray
chip.

7. The main applications of microarrays include:
Comparative genome analysis (Healthy and infected cell)
Functional genome analysis (to describe interactions
between genes at different time point)
Developing knowledge of genefunction
Discovery of drugs
Diagnostics and genetic engineering
Toxicological research (Toxicogenomics)

8. Principle of Microarray technique
• mRNA : carries the genetic information from
the cell nucleus to the cytoplasm for protein
synthesis. These mRNAs synthesize the
corresponding protein by translation.
• Indirectly by assessing the various mRNAs,
we can assess the genetic information or the
gene expression thus helps in the
understanding of various processes behind
every altered genetic expression.

9. • The principle behind microarrays is
hybridization between two DNA strands

10. Major steps in Microarray
1. Preparation of microarray
2. Preparation of labelled probes
3. Hybridization
4. Scanning, imaging and data analysis

11. PROBE TYPE ADVANTAGES DISADVANTAGES
PCR Products Inexpensive Handling problems
Hard to design to avoid
cross- hybridization
Unequal amplification
Oligos Can be designed for many
criteria
Easy to handle Normalized
concentrations
Expensive
Affymetrix GeneChip High quality data
Standardized arrays
Fast to set up
Multiple probes per gene
Expensive
Arrays available for limited
number of species
Methods for constructing thearrays:

12. • Conventional methods canbe usedto produce the
sequences (oligonucleotides), and these canthen be
printed directly ontothe microscope slide (which is
first overlaid with acoating that is positively charged).
Microspotting Techniques

15. •First usedby AGILENT
•Thisis anon-contact process.
•Minute volumes of reagentsare delivered to
defined locations on the slide similar to
‘ink-jet’ printing methods.
•A biochemical sampleis loaded into a
miniature nozzleequipped with apiezoelectric
fitting (rectangles) and anelectrical current is
usedto expel aprecise amount of liquid from
the jet onto the substrate.
PiezoelectricPrinting

16. After the first jetting
step, the jet iswashed
and asecondsampleis
loaded and deposited to
an adjacent address.A
repeated seriesof cycles
with multiple jets
enablesrapid
microarray production.

18. • Thismakesuseof semiconductortechnologies.
• This‘in situ’fabrication technique wasdevelopedby
Affymetrix,and is usedto produce theirGeneChips.
• A mercury lamp is used,activates DNAbases.
Photolithography

19. The photolithographic method
• Treat substrate with chemically protected “linker”
molecules, creating rectangular array.
• Selectively expose array sites to light deprotects
exposed molecules, activating further synthesis .
• Flush chip surface with solution of protected
A,C,G,T.
• Binding occurs at previously deprotected sites.
• Repeat steps 2&3 until desired probes are
synthesized.

20. • The mask only allows light to pass to specific
features on the chip.

21. •Achipmodified with photolabile protecting groups is selectively
activated for DNAsynthesis by shining light through a photomask.
•The chip is then flooded with aphotoprotected DNAbase,resulting in
spatially defined coupling on the chipsurface.
•Asecondphotomask is usedto deprotect defined regions of thechip.
•Repeateddeprotection and coupling cyclesenable the preparation of
high- density oligonucleotide microarrays.

22. • Principle of microarray technique
• Preparation of microarray
–Microspotting technique
–Piezoelectric printing
–Photolithography

23. DNA Microarray Technology
• High-throughput and versatile technology used
for parallel gene expression analysis for
thousands of genes of known and unknown
functions.
• Used for detection of polymorphisms and
mutations in genomic DNA
• A DNA microarray is a collection of microscopic
DNA spots on solid surface.
• Each spot contains picomoles of a specific DNA
sequence, known as probes or reporters.

24. • Each identified sequenced gene on the glass,
silicon chips or nylon membrane corresponds to a
fragment of genomic DNA, cDNAs, PCR
products or chemically synthesized
oligonucleotides and represents a single gene.
• Probe-target hybridization is usually detected and
quantified by detection of fluorophore, silver, or
chemiluminescence labelled targets to
determine relative abundance of nucleic acid
sequences in the target.

25. • The principle of DNA microarray technology
is based on the fact that complementary
sequences of DNA can be used to hybridise,
immobilised DNA molecules.
Sample
Preparation
and labelling
Washing Image
acquisition
Data Analysis

26. Sample preparation
Cy 3 Cy 5

27. Array Hybridisation
Labelled cDNA mixed together
Purification
the mixed labelled cDNA is competitively hybridised
against denatured PCR product
- cDNA molecules spotted on a glass slide

29. • Slide is dried and scanned to determine how
much labelled cDNA (probe) is bound to each
target spot.
• Hybridized target produces emissions.

30. Upregulated genes
Downregulated genes
Equal abundance

31. Types of DNA Microarray/ DNA Chips
• cDNA based microarray
• Oligonucleotide based microarray

32. cDNA based microarray
• The first type of DNA microarray technology
developed.
• This type of chips are prepared by using
cDNA, it is called cDNA chips or cDNA
microarray or probe DNA.
• The cDNAs are amplified by using PCR.
• These are immobilized on a solid support
made up of nylon filtre of glass slide.

33. • It was pioneered by Patrick Brown and his
colleagues at Stanford University.
• Produced by using a robotic device which
deposits (spots) a nanoliter of DNA onto a
coated microscopic glass slide (50-150 µm in
diameter).

34. Sample Preparation
mRNAhasbeen extracted from the cells or tissues
under study, it is converted into DNAby the useof
the reverse transcriptase enzyme.
During this reaction, the DNAis labelled by the
incorporation of fluorescent or radioactive
nucleotides into the DNA.
Thetwo samples are labelled using two different
fluorescent dyes- say,redor green.Themost
common dyesin useare Cy3(Green) and Cy5
(Red).

38. Oligonucleotide based microarray
• Often referred to as a "chip" which involves
in situ oligonucleotide synthesis.
• Gene chip (DNA chip, Affymetrix chip)
• Oligonucleotide (20~80-mer oligos) is
synthesized in situ (on-chip).
• Developed at Affymetrix, Inc. , under the
GeneChip® trademark

39. AFFYMETRIX MICROARRAY

40. Affymetrix Chip
• Each gene has 16 – 20 pairs of probes synthesized on
the chip.
• Each pairs of probes have two oligonucleotide.
• Perfect match (PM, reference seq) ATG…C…TGC
(20-25 bases).
• Mismatch (MM, one base change) ATG… T …TGC
• A MM oligo is identical to a PM oligo except that the
middle nucleotide (13 th of 25) is intentionally replaced
by its complementary nucleotide .
• The scanned result for a given gene is the average
differences between PM and MM signals, over probes.

42. A Probe Set for Measuring Expression Level of a
Particular Gene probe pair gene sequence

44. • The black features represent no intensity (no RNA hybridized
to the respective probe in the feature).
• The intensity level from lowest to highest by colour is:
Dark blue -> Blue -> Light Blue -> Green -> Yellow ->
Orange -> Red - > White .
• More intensity means more RNA bound to a specific feature,
which basically means the gene was expressed at a higher
level.

46. Affymetrix GeneChip experiment

47. Affymetrix GeneChip experiment
• Labelled cRNA randomly fragmented in to pieces anywhere
from 30 to 400 base pairs in length.
• The fragmented, Biotin-labeled cRNA is added to the array
• Anywhere on the array where a cRNA fragment and a probe
are complimentary, the cRNA hybridizes to the probes in the
feature.
• The array is then washed to remove any cRNA that is not stuck
to an array then stained with the fluorescent molecule that
sticks to Biotin (Cy5 conjugated to streptavidin).
• Lastly, the entire array is scanned with a laser and the
information is kept in a computer for quantitative analysis of
what genes were expressed and at what approximate level.

48. Procedure

51. Analysis of DNA microarray
• Bioconductor, an open source and open
development software project for the analysis
of genomic data primarily based on the R
programming language, contains a number of
program packages for microarray data
analyses and is arguably the most
comprehensive resource for such applications.
(Gentleman et al., 2004)

53. Advantages of DNA microarray
• To study the behaviour of many genessimultaneously.
• Thetechnique is very fast: there canbe asmany as150
copies of an array of 12,000 genesprinted in only 1day.
• DNAmicroarray technology is relatively cheap to use:
• the initial cost of constructing an arrayer is
approximately$60,000;
• after this, the cost per copy of amicroarray is
small, usually lessthan $100.

54. • Thetechnique of DNAmicroarrays is very user-friendly:
• the technique is neither radioactive nor toxic
• the microscope slide is a convenient base for
the technique
• arrays are cheap and easily replaced
• A major advantage of DNA microarrays is that
information about the sequence of the DNA is not
required to construct and usethe DNAmicroarrays.

55. Limitations
• Aswell asthe cost of robotics to perform the technique,
there maybe technicallimitations.
• The technique of DNA microarrays is currently limited
not by the number of probes on an array, but by the
resolution of the scanner used.
• Toomuch data all at once. Cantake quite awhile to
analyzeall the results.
• Theresults maybe too complex tointerpret
• Theresults are not always reproducible
• Theresults are not always quantitativeenough
• Thetechnology is still tooexpensive

57. Microarray in Genomics

58. • The main application of genomic microarrays is
represented by gene expression profiling.
• Basically, two types of genomic microarrays are
available:
• wide genome and focused arrays.
• Wide-genome arrays are designed to bear on them as
many genes as possible.
• Currently Affymetrix HU133 plus v.2 gene chips
have around 47 000 genes or (ESTs) on them.
• Focused arrays are designed to bear few
tens/hundreds of genes of interest.

59. • Gene expression studies: data sets can vary greatly
in size. The data heterogeneity is where the big
challenge lies for the scientist, and a crucial role is
thus played by bioinformatics and biostatistics.
• Softwares: Microarray Suite, GeneSpring, Partek
Pro.
• All software packages are equipped with visualization
tools, such as self-organizing maps, hierarchical
clustering, principal component analysis, relevance
networks, etc.

60. • Self-organizing maps: genes are plotted on a
two-dimensional graph based on their
expression level across all samples.
• Thus, groups of genes with a similar
expression pattern will have a similar trend
within the graph.

61. Hierarchical clustering analyses:
• Genes are plotted against samples with a dendrogram,
which is sort of a mock phylogenetic tree, whose
branches connect genes related by a similar expression
pattern: the shorter the branch, the stronger the
correlation.
• The dendrogram is connected with its branches to the
clustering map, where genes are represented by squares
colored in green/red or blue/yellow, based on their
differential expression level (up- or down-regulation),
which define specific molecular fingerprints.

63. • Even though visualization tools can give a
comprehensive overview of the whole study
performed, they are usually derived from the
application of filters resulting in lists of at least
hundreds of genes that might be difficult to interpret
and make a sense of, without additional statistical
analyses.
• Once a specific set of genes is identified as the
significant one, validation studies are required in
order to confirm gene expression profile
observations.

64. Such validation should be preferentially done by
• RT-PCR, quantitative PCR (Taqman),
• Northern and Western blotting,
• RNA protection assay, flow cytometry,
• mice knockout models.
All targeted to reconfirm previous gene expression
profiling data.

65. • cDNA microarrays containing ~9,000 unigenes was
used to identify 486 salt responsive expressed
sequence tags(ESTs) (representing ~450 unigenes) in
shoots of the highly salt-tolerant rice variety, Nona
Bokra (Oryza sativa L. ssp. Indica pv. Nona).

66. • This study identified a large number of candidate
functional genes that appear to be involved in salt
tolerance and further examination of these genes may
enable the molecular basis of salt tolerance to be
elucidated.
• The rice BiostarP-100s cDNA microarray (United
Gene Holdings,Ltd., PRC), containing 10,060
sequences representing ~9000 unigenes including
novel, known and control genes, was used to identify
salt-regulated genes.

67. • Gene expression was examined at three time points
after salt treatment (20min, 3h and 24h)
corresponding to early transient, intermediate and late
regulation.
• The significantly regulated genes at each time point
were selected for cluster analysis and for inclusion in
the salt-induced-microarray (SIM).

68. • The fluorescent signatures were scanned and
captured using a ScanArray4000 Standard
Biochip Scanning System.
• Data were analyzed using the GenePix Pro 3.0
software.

69. • The utilized genes were amplified by
polymerase chain reaction (PCR) of the
appropriate rice cDNA clones using T3 and T7
primers.
• After the resulting products were purified and
confirmed by direct sequencing, the fragments
were printed on slides using an OmniGrid
printer.

70. • Red : up-regulated genes
• Green: down-regulated
genes;
• Black: un-regulated genes
• Blanks: missing data.
Comparison of salt response gene expression in salt-stressed Nona
and IR28 plants using hierarchical cluster analysis

71. Advantages of genomic microarray
• One shot genome wide expression analysis.
• Rapid comparison between two states (control/diseased,
untretead/treated, and wild type/knockout).
• Exploration of new biological systems in a hypotheses
generating rather than hypotheses testing fashion.
• Identification of markers to elucidate molecular mechanisms
(signatures) underlying biological events and diseases.
• Rapid molecular disease classification for more accurate
molecular diagnostic, prognostic and targeted treatment.

72. Disadvantages
• Restricted access to the technology (experiments still
exprehensive to perform)
• Not yet approved as diagnostic tool by regulatory bodies
• Not a stand alone technique (need validation/confirmation
tests)
• Skilled technical personnel needed (including
biostatistician/bioinformatician for data analysis)
• Data derived from different platforms difficult to compare
• Data comparability difficult from one array version to the next
• Data obtained only partially used and published
• Data repositories and data sharing still not fully implemented
• Ethical and legal issues when dealing with patient samples

73. • Protein microarrays are miniaturized and parallelized
array technology approaches for protein–protein
interactions analysis and protein profiling.
• Typically, thousands of proteins are printed and
immobilized on functionalized glass slides, which
that can be simultaneously studied and analyzed in a
HT fashion, thereby offering a high potential for
characterizing the biology of a given cell of interest.
PROTEIN MICROARRAY

74. • The first report of using protein arrays for protein–
protein interaction, ligand binding and biochemical
investigations was by MacBeath and Schreiber in
2000.
• The success of each microarray-based screening
heavily depends on the library construction and
microarray fabrication.
Three key areas of protein characterizations:
1. functional annotation,
2. substrate fingerprinting,
3. ligand/inhibitor binding.

76. • The major challenge as compared to DNA
microarray is that the need to maintain the
structural integrity and physicochemical
properties of proteins, derived from its complexity
& variability (e.g. post-translational modifications,
etc.).

77. Classification of Protein Microarray
• Target microarrays,
• Reverse Phase arrays
• in situ expressed arrays.

78. Detection techniques for Protein microarray
Conventional fluorescence labeling : the use cyanine dyes
(e.g.: Cy3 and Cy5)
•Target protein array
•NAPPA protein array (Nucleic Acids Programmable Protein Arrays )
•Reverse phase microarray
Detection for suspension array technology
•Flow cytometry
•Magnetic bead based detection
•Quantum dots
•Gold Nano particles
Label free techniques
•Surface Plasmon Resonance
•Microcantilevers and Atomic force microscopy