DNA Microarrays

Dr. Abdul Wahab

Department of Microbiology
University of Karachi
 What is a Microarray?
• “Microarray” has become a general term, there are
many types now
– DNA microarrays
– Protein microarrays
– Tissue microarray
–…
• We’ll be discussing DNA microarrays
 What is a DNA Microarray

• A grid of DNA spots (probes) on a substrate used

to detect complementary sequences
• The DNA spots can be deposited by
– piezolectric (ink jet style)
– Pen
– Photolithography (Affymetrix)
• The substrate can be plastic, glass, silicon
(Affymetrix)
• RNA/DNA of interest is labelled & hybridizes with
the array
• Hybridization with probes is detected optically.
 Several types of arrays
• Spotted DNA arrays
– Developed by Pat Brown’s lab at Stanford
– PCR products of full-length genes (>100nt)
• Affymetrix gene chips
– Photolithography technology from computer
industry allows building many 25-mers
• Ink-jet microarrays from Agilent
– 25-60-mers “printed directly on glass slides
– Flexible, rapid, but expensive
 GeneChip Technology
Affymetrix Inc
Miniaturized, high density arrays of 1,300,000 DNA
oligos 1-cm by 1-cm

Manufacturing Process

Solid-phase chemical synthesis and

Photolithographic fabrication techniques employed
in semiconductor industry
 Array Fabrication Photolithography

• Light activated synthesis

• synthesize oligonucleotides on glass
slides
• 107copies per oligo in 24 x 24 um square
• Use 20 pairs of different 25-mers per gene
• Perfect match and mismatch
Array Fabrication Photolithography
 Affymetrix Microarrays
Raw image

1.28cm

50um
~107 oligonucleotides,
half perfectly match mRNA
(PM),
half have one mismatch (MM)
Raw gene expression is
intensity difference: PM - MM
Printed cDNA or Oligonucleotide Arrays
· Robotically spotted cDNAs (50mer) or Oligonucleotides (70mers) vs.
Affymetrix’s that uses 25mers

• Printed on Nylon, Plastic, or Glass surface

Steel spotting pin
Spotted arrays

chemically modified slides

384 well source plate

1 nanolitre spots
90-120 um diameter
 Building the chip
PCR amplification
Directly from colonies with SP6-T7
primers in 96-well plates

Consolidate into
384-well plates

Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)

Spot as microarray
on glass slides
 Microarray Life Cycle
Biological
Question

Data Analysis &

Modelling
Sample
Preparation

Microarray Detection

Microarray Reaction
 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
 Cartridge-based Expression
Microarrays
Involves Fluorescently tagged
biotinylated cRNA

-One chip per sample

-Uses single fluorescent dye
-More expensive

Affymetrix GeneChip Image

 Spotted cDNA and Oligo Glass
Arrays
Involves two dyes on the same slide
• Red dye-Cy5
• Green dye-Cy3
• Control and experimental
cDNA on same chip
 Pros/Cons of Different
Technologies
Spotted Arrays Affy Gene Chips
• relative cheap to make • expensive ($500 or more)
(~$10 slide) • limited types avail, no
• flexible - spot anything chance of specialized
you want chips
• Cheap so can repeat • fewer repeated
experiments many times
experiments usually
• highly variable spot
deposition • more uniform DNA
• usually have to make feaures
your own • Can buy off the shelf
• Accuracy at extremes in • Dynamic range may be
range may be less slightly better
 Purposes
• So why do we use DNA microarray?
– To measure changes in gene expression levels – two
samples’ gene expression can be compared from
different samples, such as from cells of different stages
of mitosis
– To observe genomic gains and losses. Microarray
Comparative Genomic Hybridization (CGH)
– To observe mutations in DNA
 Several types of arrays
• Spotted DNA arrays
– Developed by Pat Brown’s lab at Stanford
– PCR products of full-length genes (>100nt)
• Affymetrix gene chips
– Photolithography technology from computer
industry allows building many 25-mers
• Ink-jet microarrays from Agilent
– 25-60-mers “printed directly on glass slides
– Flexible, rapid, but expensive
 Central “Assumption” of Gene
Expression Microarrays
• The level of a given mRNA is positively correlated
with the expression of the associated protein.
– Higher mRNA levels mean higher protein
expression, lower mRNA means lower protein
expression
• Other factors:
– Protein degradation, mRNA degradation,
polyadenylation, codon preference, translation
rates, alternative splicing, translation lag…
• This is relatively obvious, but worth emphasizing
An Array Experiment
 STEP 1: Collect Samples
 This can be from a variety of organisms. We’ll use two
samples – cancerous human skin tissue & healthy
human skin tissue
 STEP 2: Isolate mRNA
• Extract the RNA from the samples. Using either a
column, or a solvent such as phenol-chloroform.

• After isolating the RNA, we need to isolate the mRNA

from the rRNA and tRNA. mRNA has a poly-A tail, so
we can use a column containing beads with poly-T tails
to bind the mRNA.

• Rinse with buffer to release the mRNA from the beads.

The buffer disrupts the pH, disrupting the hybrid
bonds.
STEP 3: Create Labelled DNA
• Add a labelling mix to the
RNA. The labelling mix
contains poly-T (oligo dT)
primers, reverse transcriptase
(to make cDNA), and
fluorescently dyed nucleotides.
• We will add cyanine 3
(fluoresces green) to the
healthy cells and cyanine 5
(fluoresces red) to the
cancerous cells.
• The primer and RT bind to the
mRNA first, then add the
fluorescently dyed nucleotides,
creating a complementary
strand of DNA
 STEP 4: Hybridization
• Apply the cDNA we
have just created to a
microarray plate.

• When comparing two

samples, apply both
samples to the same
plate.

• The ssDNA will bind to

the cDNA already
present on the plate.
Hybridization chamber
3XSSC

HYB CHAMBER

ARRAY

LIFTERSLIP

SLIDE
LABEL
SLIDE LABEL

• Humidity
• Temperature
• Formamide
(Lowers the Tm)
STEP 5: LASERS!
 STEP 5: Microarray Scanner
 The scanner has a laser, a computer,
and a camera.

 The laser causes the hybrid bonds to

fluoresce.

 The camera records the images

produced when the laser scans the
plate.

 The computer allows us to

immediately view our results and it
also stores our data.
Scan
Scan

Green: down regulate

Red: up regulate
Yellow: equal level
 STEP 6: Analyze the Data
 GREEN – the healthy sample hybridized
more than the diseased sample.
 RED – the diseased/cancerous sample
hybridized more than the non diseased
sample.
 YELLOW - both samples hybridized
equally to the target DNA.
 BLACK - areas where neither sample
hybridized to the target DNA.
 By comparing the differences in gene
expression between the two samples, we
can understand more about the
genomics of a disease.
Image Analysis & Data Visualization
Cy5 Cy5
log2
Cy3 Cy5 Cy3 Cy3

200 10000 50.00 5.64

4800 4800 1.00 0.00
9000 300 0.03 -4.91

Experiments
Underexpressed 8
4
2
Genes

fold
2
4
Overexpressed
8
 Data Normalization
• Normalize data to correct for variances
– Dye bias
– Location bias
– Intensity bias
– Pin bias
– Slide bias
• Control vs. non-control spots
 Data Normalization
Uncalibrated, red light under Calibrated, red and green equally
detected detected
 Hierarchical clustering
Genomic Reprogramming in Response to Oxidant minutes
0 10 20 40 60 120

One-third of genome expression is

transiently reprogrammed

6218 genes

Fold repression Fold induction

>9 >6 >3 1:1 >3 >6 >9
Heat map representing the expression level of selected genes of Salmonella wild type and baeR mutant in LB
+/−20mM sodium tungstate.

Appia-Ayme C, Patrick E, J. Sullivan M, Alston MJ, et al. (2011) Novel Inducers of the Envelope Stress Response BaeSR in Salmonella
Typhimurium: BaeR Is Critically Required for Tungstate Waste Disposal. PLoS ONE 6(8): e23713. doi:10.1371/journal.pone.0023713
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023713
Differentially expressed genes during cell cycle
 Microarray Limitations
• Cross-hybridization of sequences with high identity
• Chip to chip variation
• True measure of abundance?
• Does mRNA levels reflect protein levels?
• Generally, do not “prove” new biology - simply
suggest genes involved in a process, a hypothesis
that will require traditional experimental
verification.
• What fold change has biological relevance?
• Need cloned EST or some sequence knowledge --
rare messages may be undetected
• Expensive!! Not every lab can afford experiment
repeat.
• The real limitation is Bioinformatics
Microarray Potential Applications
• Biological discovery
– new and better molecular diagnostics
– new molecular targets for therapy
– finding and refining biological pathways
– Mutation and polymorphism detection

• Recent examples
– molecular diagnosis of leukemia, breast
cancer, ...
– appropriate treatment for genetic signature
– potential new drug targets