Lecture -5

Transcriptomics Analysis through

DNA Microarray
Course: Trends in Nucleic acid Sequencing Techniques (MBFS 518)

Dr Farheena Iqbal Awan

Assistant Professor
CAMB
• Microarrays allow simultaneous measurement of thousands of genes.
• Based on nucleic acid hybridization principles.

WHY Microarray
• Used in genomics, transcriptomics, diagnostics.
• Simultaneous analysis of thousands of genes.
• Discovery of gene functions.
• Genome-wide network analysis.
• Analysis of mutants and transgenics.
• Identification of drug targets.
• Causal understanding of diseases.
• Clinical studies and field trials
 Principle of Microarray analysis
• The microarray are solid supports, usually of
glass or silicon, upon which immobilized
nucleic acid sequences (called probe) are
attached in pre-determined grid fashion.
• The size of the spots or target is usually less
than two hundred micrometers in diameter.
• On a glass slide up to tens of thousands of
spots can be arrayed in a total area of a few
square centimeters
• Target: one or more labelled nucleic acid
samples obtain from any source. That are
hybridized with microarray.
• Scanner or detection system that quantities
the hybridization signals.
 Mechanism of Microarray analysis

• Target labelling: The mRNA or DNA sample that is being

analyzed, is converted to a labelled population of nucleic
acids.
• Fluorescent dyes, most commonly Cyanine dyes Cy3 and
Cy5, are used for labelling in microarray analysis.
• Hybridization: the labeled nucleic acid from two analyzed
samples are placed in the microarray so that each target
gets hybridized to its complementary probe strand through
hydrogen bonding.
• The non-specific bonding sequences while remain
unattached and washed out during the washing step of the
process.
 Mechanism of Microarray analysis

• The binding of the fluorescently labeled target

sequences to a probe sequence on array generate a
signal.
• Two or more samples labelled with different fluorescent
dyes can be hybridized simultaneously, resulting in
simultaneous hybridization taking place at each target
spot.
• The total strength of the signal reflect the number of
target bind to that probe.
 Application of Microarray analysis
Fig. Dual color differential microarray
analysis.
Dual color microarray hybridization
red = Cy5 dye signals
green = Cy3 dye signals
If equal signal is obtained from a spot, it will
appear yellow.
Shades of green and red denote differences in
relative abundance in one or the other
sample.
By measuring the different fluorescent signals
associated with each spot, the Relative abundance
(not absolute) of specific sequences in each of the
samples can be determined
• Scanning and data analysis:
Microarray scanners typically contain two different
lasers that emit light at wavelengths that are
suitable for exciting the fluorescent dyes used as
labels.
A confocal microscope attached to a detector
system records the emitted light from each of the
microarray spots.
Specialized software extracts primary data from
scanned microarray slide images, normalizes this
data to remove the influence of experimental
variation, and finally manipulates the data so that
biologically meaningful conclusions can be made.
 Application of Microarray analysis
1- Gene expression analysis
• The predominate application of DNA microarrays has been to measure gene expression levels in two
samples.
• One of these samples is a control and the other is derived from cells whose response or status is
being investigated.
• In this application, RNA is extracted from the cells of interest and either converted to labelled cRNA
or converted to a labeled cDNA or.
• A wide variety of methods have been developed for labeling of the cDNA or cRNA including:
✓ incorporation of fluorescently labeled nucleotides during the synthesis,
✓ incorporation of biotin labeled nucleotide which is subsequently stained fluorescently labeled streptavidin,
✓ incorporation of a modified reactive nucleotide to which a fluorescent tag is added later.
• Each of these samples is labelled with a different fluorescent dye, and equal amounts of the each
labelled samples are combined and hybridized with the microarray.
• The fluorescent signals corresponding to the two dyes are measured independently from each spot
after hybridization.
• After normalization, the intensity of the two hybridization signals can be compared.
• Equal signal from both samples suggests equal expression in both samples
• Microarray analysis does not give information about absolute gene expression levels
in the samples. This is because the intensity of the fluorescent signals is not only
proportional to the number of hybridized fragments but also to the length of these
fragments and the number of fluorescent labels each fragment carries, i.e. labelling
density.
• A strong hybridization signal from microarray analysis does not necessarily
correspond to a highly expressed gene; it could be derived, for example, from a gene
that is expressed at a relatively low level but yields long, highly-labelled probe
fragments.
Gene expression analysis with microarrays has been applied to numerous mammalian
tissues, plants, yeast, and bacteria alike.
These studies have examined :
➢The effects of treating cells with chemicals,
➢The consequences of over-expression of regulatory factors in transfected cells
➢ Compared mutant strains with parental strains to delineate functional pathways.
➢ In cancer research microarrays have been used to find gene expression changes in
transformed cells and metastases, to identify diagnostic markers, and to classify
tumors based on their gene expression profiles
 Application of Microarray analysis
2- Transcriptional factor binding analysis
• Gene regulation mechanism is also studied by elucidation of transcription factors - gene
interactions. One of the most common microarray-based approaches for this is ChIP-chip
(Chromatin Immunoprecipitation followed by microarray analysis).
• Crosslinking: Cells are treated with formaldehyde to crosslink DNA and associated
proteins (like TFs), fixing the protein-DNA interactions in place.
• Chromatin Extraction and Shearing: DNA is sheared via sonication or enzymatic
digestion) to generate small DNA fragments (~200–1000 bp).
• Immunoprecipitation (IP): An antibody specific to the transcription factor is used to
immunoprecipitate the TF-DNA complexes. This step pulls down only those DNA
fragments that are bound to the TF of interest. The antibodies may be attached to a solid
surface, may have a magnetic bead, or some other physical property that allows separation
of cross-linked complexes and unbound fragments. This can be done either by using a
tagged protein with an antibody against the tag (ex. FLAG, HA, c-myc) or with an antibody
to the native protein.
• Crosslink Reversal and DNA Purification:
Crosslinks are reversed (usually by heating),
and the DNA is purified. You now
have a population of DNA fragments
representing TF-bound sites.
• After an amplification and
denaturation step, the single-stranded
DNA fragments are labeled with a
fluorescent tag such as Cy5 or Alexa
647.
• Finally, the fragments are poured
over the surface of the DNA
microarray, which is spotted with
short, single-stranded sequences
(usually promotor and regulatory
regions). Whenever a labeled
fragment "finds" a complementary
fragment on the array, they will
hybridize and form again a double-
stranded DNA fragment
• This technique is commonly referred
to as “ChIP-chip” for Chromatin
Immuno-Precipitation on a “chip”
or microarray.
 Application of Microarray analysis
3- Genotyping
Microarrays have been widely used as single-nucleotide-polymorphism (SNP) genotyping
platforms.
Several alternative approaches have been used to detect SNP’s but the most commonly
used are
1. Allele discrimination by hybridization as used by Affymetrix (Wang et al., 1998),
2. Allele specific extension and ligation to a “bar-code” oligo which is hybridized to a
universal array (the Illumina “Golden Gate Assay”(Fan et al., 2003))
3. Approaches in which the arrayed DNA is extended across the SNP in a single nucleotide
extension reaction (the Arrayed Primer Extension assay or the Infinium Assay of
Illumina).
SNP detections strategies.
A) 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 effect on hybridization. Typically, multiple
array positions are used for each allele to improve signal to noise.
Experimental Workflow:

• High-quality genomic DNA is extracted from the sample (e.g., blood, cell lines).
• Genomic DNA is digested using restriction enzymes (NspI and StyI).
• These enzymes recognize specific 6-base pair sequences (NspI: 5′-RCATGY-3′ and StyI: 5′-
CCWWGG-3′) and cut the DNA at those sites, generating reproducible DNA fragment ends.
• The digested DNA fragments typically range from 200–1100 bp.
• Adapters (double stranded oligonucleotide) are ligated to the digested fragments.
• A sticky end that is complementary to the overhang left by the restriction enzyme.
• A blunt or double-stranded region with a known sequence to serve as a PCR priming site.
• T4 DNA ligase is used to catalyze the ligation reaction.
• Adapter-ligated fragments are amplified using universal primers through PCR.
• PCR products are fragmented to ~25–125 bp through DNase I.
Fragments are labelled with biotin.
Key Reaction:
TdT + fragmented DNA + biotin-ddUTP → DNA with biotin on the 3′ end
(TdT) -Terminal deoxynucleotidyl transferase: dd nucleotides without a template, so it
efficiently tags the 3′ ends of all DNA fragments.
biotin-ddUTP - biotinylated dideoxynucleotides
The biotin-labeled DNA is hybridized to the microarray containing millions of probes.
After washing, the array is incubated with streptavidin–phycoerythrin (SAPE), which binds
to the biotin.
Scanned using a high-resolution scanner.
Raw intensity data is processed using software (e.g., Genotyping Console, Chromosome
Analysis Suite, or third-party tools).
Allele calling, CNV detection, and LOH analysis are performed.
Example Applications of Affymetrix scans.
1. Genome-Wide Association Studies (GWAS) for SNP associated with disease.
Example: contains more than 906,600 single nucleotide polymorphisms (SNPs) and
more than 946,000 probes for the detection of copy number variation.
To study genetic risk factors for Type 2 Diabetes, Autism Spectrum Disorder in different
populations.
2. Copy Number Variation (CNV) Detection - The Affymetrix CytoScan HD Array is a
high-resolution, genome-wide microarray specifically designed for detecting copy
number variations (CNVs) and loss of heterozygosity (LOH) associated with constitutional
genetic disorders, developmental delays, intellectual disability, and cancer.
3. Pharmacogenomics: Affymetrix DMET (Drug Metabolizing Enzymes and
Transporters) array Identify SNPs in drug metabolism genes (e.g., CYP450) to predict drug
response, adverse effects, dosage adjustment etc.
4. Population Genetics: Affymetrix Genome-Wide Human SNP Array 6.0 Assessing
genetic diversity and population structure using dense SNP data across individuals from
different ethnic backgrounds
B) Illumina’s “Golden Gate Assay”-
Two Allele Specific Oligos (ASO) are each tailed with a different universal primer (1 and 2) are
hybridized in solution to genomic DNA.
A third Locus Specific Oligo (LSO) that is complementary to the same locus is tailed with a
“barcode” sequence and a third universal primer (3).
If an ASO perfectly matches the SNP allele, a DNA polymerase extends the ASO by one base,
bridging the gap.
A ligase then joins the ASO to the LSO.
PCR is performed using primers complimentary to universal sequences 1, 2 and 3.
The PCR primers complimentary to the
universal sequences 1 and 2 are
labeled with a unique fluorophore
(Cy3 and Cy5).
PCR products are hybridized to a universal BeadChip array, which contains complementary
oligos to the barcode sequence on the third oligo.
Each bead type corresponds to a specific SNP.
The use of multiple barcodes (one for each locus of interest) allows the assay to be
multiplexed to sample many loci.
Available for 96 to 1536 SNPs.
C) Arrayed primer extension (APEX) –
A multiplexed genotyping technique in which short
oligonucleotide primers are immobilized (at 5’ end) on a solid
surface (e.g., glass slide or microarray chip), and DNA
polymerase extends the primer by one fluorescently labeled
nucleotide complementary to the template.
Genomic DNA is fragmented, denatured and hybridized to the
array.
The oligo on the array is extended in single nucleotide dye
terminator sequencing reaction.
After the extension step, the slide is washed and scanned with a
fluorescence scanner.
Each color corresponds to one of the four bases, so the
incorporated nucleotide at each primer location reveals the
genotype or mutation status.
D) Illumina’s Infinium assay – This assay is similar to the APEX assay except that the oligo
to be extended is on a bead and the single nucleotide that is added is labeled with a
nucleotide specific hapten as opposed to a fluorophore.
The haptens are then detected by staining with fluorescently labeled proteins that bind
each hapten.
 Results Validations
For gene expression microarrays, the hybridization signal is influenced by a number of
variable factors:

➢Including the number of specific transcripts in the labelled samples,

➢The labelling method,

➢the properties of microarray printer pens,

➢Hybridization conditions, and slide surface chemistry.

Furthermore, variation in microarray signal is observed not only between different slides but
also between different replica targets spotted onto different locations of the microarray slide.
 Positive Control targets
Different strategies have been devised for microarray control purposes.

Internal Spike-in Controls: Pooled genomic DNA of some other organism (e.g., bacterial
RNAs in a human microarray).
• Check for labelling and hybridization
Housekeeping Genes: Endogenous genes like GAPDH, ACTB, or 18S rRNA are expected
to be expressed consistently across conditions.
Check for labelling and hybridization
Control Spots on the Array: Some microarray platforms have spots pre-printed with
control probes (e.g., for hybridization or dye balance).
• These are not part of your sample preparation but help validate sample prep indirectly
through the signal pattern.
 Negative Control targets

Negative Control: No-Template Control (NTC), A mock sample (water or buffer) is

processed like a real sample through extraction and labeling.
• Confirms absence of contamination or non-specific labeling.
Negative Spike-in Controls: Synthetic sequences added that should not hybridize to any
probe.
• If they show signal, it indicates non-specific binding or background noise.
Array-Included Negative Probes: Some arrays include probes with random sequences or
known non-target sequences.
• They should show no hybridization—used to estimate background signal.
 Advantages and Limitations
• Advantages: High-throughput, economical
• Limitations: Requires known sequences,
cross-hybridization

• RNA-seq offers higher resolution and is

replacing microarrays