DNA Microarrays

Dr Divya Gupta
 What are DNA microarrays
• It is a collection of microscopic DNA spots attached to a solid surface.

• The principle of DNA microarray technology is based on the fact that

complementary sequences of DNA can be used to hybridise immobilised
DNA molecules

• Genome chip, DNA chip or gene array: Traditional solid-phase array:

collection of orderly microscopic spots or specific probes attached to a
solid surface (glass, plastic or silicon biochip)

• Bead array : It is a collection of microscopic polystyrene beads, each with

a specific probe and a ratio of two or more dyes, which do not interfere
with the fluorescent dyes used on the target sequence.
Methodology of DNA Microarrays

•Printing of Microarrays
•Isolation of RNA
•Synthesis of fluorescently labeled probe (Cy-5, Cy-3)
•Hybridization of the probe
•Scanning of Microarrays
•Data analysis and interpretation of data
Methodology of DNA Microarrays
Making of Microarrays

Automatic microarray printing machine

DNA microarray printing
 Gene Array making

•Usually 25 mers, are directly synthesized onto a glass wafer by a combination of

semiconductor-based photolithography and solid phase chemical synthesis technologies

•Each array contains up to 400,000 different oligos and each oligo is present in millions of
copies

•Oligonucleotide probes are synthesized in known locations on the array

Genes are represented by Oligonucleotides on the array
•Each gene is represented on the array by a series of different oligonucleotide
probes

• Each probe pair consists of

Perfect match oligonucleotide: a sequence exactly complimentary to the
particular gene and thus measures the expression of the gene
The mismatch oligonucleotide: differs from the perfect match probe by a single
base substitution at the center base position, disturbing the binding of the target

gene transcript

•Helps in determination of the background and nonspecific hybridization that

contributes to the signal measured for the perfect match oligo

•Software subtracts the hybridization intensities of the mismatch probes from those
of the perfect match probes to determine the absolute or specific intensity value for
each probe set

• Probes are chosen based on current information from Genbank and other
nucleotide repositories
 RNA Extraction
• RNA can be extracted from tissue or cultured cells using
standard protocols
Amount of mRNA required is about 0.5μg ~to 20/μ/g of total
RNA,
• If amount of mRNA (or DNA) is scarce an amplification step is
needed before labeling
• Quality of mRNA is checked by Bioanalyzer or running a
agarose gel
RNA extraction and quality check

28s
18s
Quality check of RNA by Bioanalyzer
 Synthesis of labeled cDNA
• mRNA is retro-transcribed using reverse transcriptase to
generate cDNA.
• Labeling is achieved by including in the reaction (or in a
separate reaction-post labeling) modified fluorescent
nucleotides that are made fluorescent by excitation at
appropriate wavelengths.
• The most common fluorescent dyes used are Cy3 (green) and
Cy5 (red).
• The unincorporated dyes usually are removed by column
chromatography or ethanol precipitation
 Hybridization

• Hybridization is carried out according to conventional protocols

• Hybridization solution contains saline sodium citrate (SSC), sodium
dodecyl sulphate (SDS) as detergent, non-specific DNA such as yeast DNA,
salmon sperm DNA, or repetitive sequences, blocking reagents like bovine
serum albumin (BSA) or Denhardt’s reagent, and labeled cDNA from the
samples
• Hybridization temperatures range from 42°C to 45°C for
cDNA-based microarrays and from 42°C to 50°C for oligo-based
microarrays.
• Hybridization volumes vary between20/μ/L to 1 mL depending on the
microarray technology.
• A hybridization chamber is usually needed to keep temperature and
humidity constant.
 Scanning
• After hybridization, the microarray is washed in salt buffers of decreasing
concentration, dried by slide centrifugation
• Then the slide is read by a scanner which consists of a device similar to a
fluorescence microscope coupled with a laser, robotics, and digital camera to
record the fluorescent excitation.
• The robotics focuses on the slide, lens, camera, and laser by rows similar to a
common desktop scanner.
• The amount of signal (color) detected is presumed to be proportional to the
amount of dye at each spot in the microarray and hence proportional to the
RNA concentration of the complementary sequence in the sample.
• The output is, for each fluorescent dye, a monochromatic (non-colored)
digital image file typically in TIFF format. False color images (red, green, and
yellow) are reconstructed by specialized software for visualization purposes
only.
Microarray
 Image analysis
• The digital images are loaded in specialized software with a pre-loaded
design of the microarray (grid layout) which instructs the software to
consider number, position, shape, and dimension of each spot.

• The grid is then accommodated to the actual image automatically or

manually. Fine tuning of spot positions and shapes is usually performed to
avoid any bias in the robotic construction of the microarray.

• Human involvement is needed to mark those spots that could be artifacts

such as bubbles or scratches which are common. Finally, an automated
integration function is performed using the software to convert the actual
spot readings to a numerical value
 Image analysis

• The integration function considers the signal and background noise

for each spot. The output of the image analysis may be commonly
a tab-delimited text file or a specific file format. This process varies
from automatic or semi-automatic to manual depending on type of
microarray used

• Common image analysis software are

• ScanArray (PerkinElmer, Waltham, MA, USA),
• GenePix (Axon),(Molecular Devices Corporation, Union City, CA, USA)
• TIGR-SpotFinder/TM4(www.tigr.org), (The Institute for Genomic Research,
Rockville, MD, USA)
• GeneChip (Affymetrix, Santa Clara, CA, USA).
 Normalization

• Systematic errors are introduced in labeling, hybridization, and

scanning procedures

• The main aims of normalization is to correct for these errors

preserving the biological information and to generate values that
can be compared between experiments, especially when they were
generated in, and with, different times, places, reagents,
microarrays, or technicians

• There are two types of normalization

“within” and “between” array normalization.
“Within” array normalization refers to normalization applied in the
same slide and it is applicable, generally, to two-dye technologies
 Normalization
• M = Log2(R/G) and A = Log2(R*G) /2 where R and G are the red and
green readings respectively. Under the assumption that the majority of
genes have not been differentially expressed, the majority of the M
Values should oscillate around zero.

• “Within” normalization is finally performed shifting the imaginary line

produced by the values of M (in vertical axis) to zero along the values of
A (in horizontal axis).
It is, usually is performed by spatial blocks to avoid any bias in the
microarray printing process (called print-tip-loess).

• “Between” normalization is necessary when at least two slides are

analyzed to guarantee that both slides are measured in the same scale
and that its values are independent from the parameters used to generate the
measurements
 Filtering
• A common practice is to remove genes that have
not shown significant changes across samples,
• Genes with several missing data, or those
whose average expression is very low
 Transformation

• The numerical values from image analysis are commonly integer

numbers between one and 32,000 for both signal and
background
• The background normally is subtracted from the signal.
• The distribution of these values is, however, concentrated in a
narrow range and, therefore, is transformed using logarithms
(base 2 generally) which generate normal-like distributions.
Statistical Analysis
Scatter plot analysis by BRB array tools

Upregulated genes or down regulated genes(>2folds) are represented by red dots

Black dots represent genes which are not significantly affected
Microarray comparison of developmental changes in gene expression

Within each Venn area, the number of increased transcripts is shown in red and the number
of decreased transcripts in green
Values in parentheses give the number of transcripts modulated in the Klf4CN versus WT
comparison.

Invest Ophthalmol Vis Sci. 2011 Jul; 52(8): 4951–4962.

Categorization of the affected transcripts based on biological functions

Panther analysis tool (www.pantherdb.org)

Corresponding gene ontology (GO) terms are shown in parentheses in the legend
Most Significantly Affected Transcription Factor Families

Increased during Decreased during

Transcription Factor
Family Conjunctival Conjunctival
Development Development
Krüppel-like factors Klf4, Klf6, Klf9 Klf13, Klf7, Klf10, Gli2
Epithelial-specific Ets
Spdef, Ehf, Elf3, Elf5 Ets1
(ESE) factors
Forkhead box family Foxa3, Foxa1, Foxk1,
Foxp2
proteins Foxo3, Foxp1
High-mobility group Hmgb1, Hmgb2 Hmg20b
proteins
Sox family members Sox21 Sox4, Sox11, Sox12
Interferon regulatory Irf1 Irf3
factors
Heat Map of microarray data
Biomarker Detection. Larger differences in gene expression are more likely
to be genuine differences between two groups of samples (A and B) than
small differences. In this case, a large number of samples are more
informative than individual replications.
Validation of microarray data by Real-time PCR

Invest Ophthalmol Vis Sci. 2011 Jul; 52(8): 4951–4962.

 Detection of SNPs Using Microarrays

•Allele discrimination by hybridization – Oligos that are complimentary to each allele are
placed on the array and labeled genomic DNA is hybridized to the array

• The variant position is placed in the center of the oligo (typically 25bp on Affymetrix
arrays) as this position has the greatest affect on hybridization
 Advantages of Microarrays

the expression of thousands genes at the same time

a lot of results fast

Compare the activity of many genes in diseased and

healthy cells

categorize diseases into subgroups

 Disadvantages of Microarrays
•Too much data all at once. Can take quite a while
to analyze all the results.

•Results may be too complex to interpret

•Results are not always reproducible

•Results are not always quantitative enough

• The technology is still too expensive

 Application of Microarrays

•Gene expression profiling

•Comparative genomic hybridization

•SNP detection

•Tiling array