Monitoring Gene Expression:

an introduction to
microarrays and quantitative
PCR

Janine Kirby

GEM6410
 Learning objectives:
• Introduction to microarrays
• Types of arrays available
• Advantages and disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
• Introduction to microarrays
• Types of arrays available
• Advantages and disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 Microarray Analysis /
Transcriptomics
• the study of the complete set of
RNA transcripts present in the cell
at any one time

• mRNAs provide a snapshot of the

genome’s plans for protein
synthesis under the cellular
conditions at that moment
 Analysing Gene
Expression
• Northern blot
• DNA used to probe RNA
• Requires 10ug RNA, result in 4-7 days
• Reverse transcriptase (RT) PCR
• RNA transcribed to cDNA, cDNA amplified by
PCR
• 1-2ug RNA, result in 1 day, not quantitative
• Quantitative PCR
• Quantitative RT-PCR, product level detected
throughout PCR
• Microarrays
• Expression analysis of thousands of genes
 Microarrays
• Function
– Quantitative detection of thousands of
transcripts present in a complex sample
• Principles
– Labelled sample is hybridised to
immobilised DNA probes of known
sequence
– Measurement of the level of bound
sample corresponds to level of
expression
 Microarray Timeline
• 1991 – First publication in Science
• 1992 – First patent of technology
• 1993 – Affymetrix created
• 1996 – Commercially available arrays
• 1998 – 21 microarray papers in
PubMed
• 2001 – 834 papers in PubMed
• 2005 – 4453 papers in PubMed
 Types of Microarrays
• Macroarrays
• cDNA sequences spotted onto nylon arrays

• cDNA arrays
• cDNA sequences spotted onto glass slides

• Oligonucleotide arrays
• Oligos spotted onto glass slides or
synthesised onto a GeneChip
 Macroarrays
• cDNA sequences spotted onto nylon
membrane –
– Commercially manufactured and homemade
– Small amounts RNA required >50ng
– Don’t require expensive equipment

– Fewer probes on membrane (4,800 Atlas

Arrays)
– One sample hybridised at a time
– Uses radioactivity/ECL = less sensitive
 cDNA Arrays
• 20,000 cDNA sequences 0.5 – 1kb
spotted onto glass slides

• Advantages
– More probes analysed
– Competing dyes used (Cy3, Cy5) and ratio
of green:red fluorescence indicative of
transcript abundance
– More sensitive than macroarrays
cDNA Arrays
 cDNA Arrays
• Disadvantages
– Expensive equipment required to analyse
the results
– Requires dye-swap experiments
– More RNA required
 Oligonucleotide Arrays
• Oligonucleotides (25 – 60mer)
synthesised directly onto glass
• Affymetrix – photolithography
• Agilent – ink-jet process
– Over 40,000 transcripts sampled
– High quality control, low variability in arrays
– High sensitivity

– Single sample per Chip

– Expensive equipment required
– 10ug RNA required
• Introduction to
microarrays
• Types of arrays available
• Advantages and
disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 Affymetrix GeneChips

• High
specificity
and
7cm
sensitivity
• Quantitative
4cm
• Reproducibl
e
 GeneChip Manufacture
Combines two technologies:
• Combinatorial chemistry
– 25-mer oligos using 4 bases (A,C,G,T)
created in 100 steps
• Photolithography
– Light is used to specify where bases
are added to the chip
 Photolithography

Array coated with light sensitive compound that

prevents nucleotides coupling.
Mask synthesised so light penetrates specific sites
on array.
Coupling able to occur between array and
Photolithography
100 reactions
required to
synthesise
25-mer oligo

Mask specific
for each of
the 100
reactions

50-400 chips
synthesised
on one wafer
• Introduction to
microarrays
• Types of arrays available
• Advantages and
disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 GeneChip Characteristics
• Each probe is an oligomer of 25 bases
• Probes come in pairs: perfect match
(PM) and a mismatch (MM) probe
– The 13th nucleotide in the mismatch
probe is non-complementary
– Mismatch probes control for hybridisation
specificity
• Each transcript represented by 11
probe pairs
 Perfect Match and Mismatch Probes

mRNA reference sequence

5´ 3´

Spaced probe sequences

Reference sequence
···TGTGATGGTGGGAATGGGTCAGAAGGACTCCTATGTGGGTGACGAGGCC···
TTACCCAGTCTTCCTGAGGATACAC Perfect Match Oligo
TTACCCAGTCTTGCTGAGGATACAC Mismatch Oligo

Perfect match probe cells

Fluorescence
intensity image

Mismatch probe cells

 Probe Design and
Selection
• Use latest databases Unigene,
Genbank, dbEST and RefSeq to identify
transcripts
• Compile multiple alignments to
determine if any DNA sequences are
alternatively spliced
• Design probes to last 600nt of cDNA
• Ensure minimal cross hybridisation
between probes
• Give good hybridisation signal intensity
 Expression GeneChips
Available
• Arabidopsis •Plasmodium
• B.subtilis •Porcine
• Barley •Rat
• Bovine •Rice
• C.elegans •Soybean
• Canine •Sugar cane
• Chicken •Tomato
• Drosophila •Wheat
• E.coli •Xenopus
• Human •Yeast
• Maize •Zebrafish
• Mouse •Nimble & Custom arrays
Human Genome GeneChip
• Human Genome U133 Plus 2.0
Array
– Genome wide expression on a single
array
• 47,400 transcripts and variants, including
38,500 well-characterised genes * *
• Probe sets, each comprising 11 *probe
* *
pairs, scattered throughout the chip
Millions
• Feature size is of identical
11um 11µm
probes / feature
11µm
 Future GeneChips
• Exon arrays – Whole genome assay
• Upto 60% genes alternatively spliced

• 5um feature size

• 4 probes per exon, each 25-mer oligo
• 1.4 million probe sets, 5.6 million probes
*
*

• Able to determine presence of

alternatively spliced transcripts
• Introduction to
microarrays
• Types of arrays available
• Advantages and
disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 Experimental Design
•MOST IMPORTANT: Good
experimental design is essential for
achieving maximum information of
statistical significance
•Considerations:
– Number of biological replicates
» 3 replicates for low variability samples e.g. cell lines
* *
» 5-6 replicates for inbred animal models e.g. Tg mice
» >10 replicates for high variability samples e.g.
humans
– Implications of pooling samples
» Small amounts of material available – pool or
amplify
Experimental Protocol
RNA is extracted

Double stranded
cDNA is synthesised

Biotin labelled copy

RNA (cRNA) is
transcribed from
cDNA

Sample is hybridised
to Genechip

Chip washed, stained

and scanned
 Labelling
Protocol
•First strand cDNA
synthesised from oligo dT
primer using reverse
transcriptase

•Second strand
synthesised from T7
primer using DNA
polymerase

•cRNA transcribed using

T7 polymerase,
 Hybridisation

• Samples added to GeneChip and hybridised for 18

hours at 42oC
• GeneChip washed in Fluidics system, then stained with
fluorescent avidin which binds biotin and amplifies the
hybridisation signal
• The GeneChip is then scanned and the results checked
on the computer
 Analysis
• Initial Analysis
– Use MAS5.0 (Microarray Analysis
Software)
– QC on hybridisation and scanning
– Creates data table of hybridisation signals
– All genes listed with calculated signal intensities
– Creates experimental report file
– % genes present, marginal, absent

– Provides snap shot of all genes expressed

in the sample when RNA was extracted
 Comparative Analysis
• How can this information can be
used?
– Comparing disease v non-disease
samples
– Comparing treated v untreated samples
– Comparing gene changes over time

– Discussed further in next lecture

 MGED and MIAME
• 1999 Microarray Gene Expression Data
(MGED) group set up
• Framework for describing microarray experiments
• Standard format for data exchange

• 2001 Minimum Information About

Microarray Experiment (MIAME) framework
proposed
• Nature Genetics 29:365-371; Nature 415:946 (2002)
• Experimental design, samples used, extraction,
labelling and hybridisation protocols used, array
design and format
 Gene Expression
Databases
• Array Express at EBI
– http://www.ebi.ac.uk/arrayexpress

• Gene Expression Omnibus

– http://www.ncbi.nlm.nih.gov/geo

• Stanford Microarray Database

– http://genome-www.stanford.edu/microarray
Microarray Manufacturers
• Affymetrix – oligos on GeneChip
• Agilent – oligos on glass
• Ambion – membrane & glass cDNA arrays
• Applied Biosystems – oligos on glass
• Clontech – membrane cDNA arrays
• Illumina – glass cDNA arrays
• Sigma Genosys – membrane cDNA arrays
• And others
• Introduction to
microarrays
• Types of arrays available
• Advantages and
disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 Quantitative PCR (Q-PCR)
• Q-PCR monitors the amplification of a
PCR product in real-time

• Q-PCR allows initial starting template

amount to be compared between
samples

• Q-PCR is sensitive, quick, reproducible,

and allows high throughput of samples
 PCR
[DNA]

Limit of
detection

Cycle
number
Principles of Real-Time Q-
PCR
• Amount of amplified product
corresponds to fluorescence
intensity
• Fluorescence levels measured at
end of each cycle
• The threshold cycle (Ct) is the first
cycle at which the fluorescence
measured is above background
 Q-PCR

• The Ct value
[DNA]

4 directly correlates
with starting
2
target
1 concentration

Cycle
threshold
(Ct)
Cycle
number
 Visualising DNA
• Fluorescent signals can be
detected using:
– Non-specific DNA binding dye
• SYBR Green I
– Seqence specific probe with
fluorescent dye and quencher
• Taqman probes
• Molecular Beacons
• Others
 SYBR Green I
• Binds to double stranded DNA
• Pros
– Cost effective, easy to use
– Ideal for optimisation of primers
– Good for high throughput of lots of
genes
• Cons
– Non-specific binding, so ensure no
primer dimers
– Cannot multiplex PCR reactions
– Less sensitive than other methods
 Taqman Probes
• Sequence specific probe with
fluorescent dye (FAM) and quencher
(TAMRA)
• Probe binds to complementary
sequence

www.gene-quantification.de/chemistry
 Taqman Probes
• As amplification occurs, DNA
cleaved by nuclease activity
• Dye no longer quenched and
fluorescence detected

www.gene-quantification.de/chemistry
 Taqman Probes
• Pros
– Sequence specific
– Can multiplex gene of interest and control

• Cons
– More expensive
– Primers and probes more difficult to
design
– Primer Express (ABI)
– Beacon Designer (Premier Biosoft)
 Molecular Beacons
• Stem-loop structure
• Central loop complementary
to target
• Arms complementary to each
other
• No target
• stem loop formation, dye and
quencher in close proximity
and no fluorescence
• Target present
• Preferentially hybridises to
target, dye separate from
quencher and fluoresence
www.gene-quantification.de/chemistry
 Molecular Beacons
• Pros
• High specificity – ideal for allele
discrimination
• Use 3-cycle PCR profile

• Cons
• Careful primer design required
• Optimum annealing temperature required
• Introduction to
microarrays
• Types of arrays available
• Advantages and
disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 Q-PCR Experimental
Protocol
• All reactions carried out in triplicate
• Optimise primer concentrations
• 50-900nM range, forward/reverse
– 50-300nM for SYBR Green
– 50-600nM for Taqman and Molecular Beacons
• Choose primer concentrations
– Lowest Ct value & Highest dRn last value
– Single PCR product
– Lowest overall primer concentration
• Optimise probe concentrations
• 50-300nM for Taqman & Molecular
Beacons
• Choose probe concentration (as above)
 Q-PCR: Quantifying
template
• To quantify starting amount of
template:
• Absolute quantification
– Dilution series of known concentration
– Generate standard curve on plate
– Samples compared to standard curve
• Relative quantification
– Standard curve to ensure PCR efficient
– Ct value of gene of interest (GOI) compared back
to control (calibrator)
– Difference between the two is the fold-change
– Also normalise GOI to control gene
 Expt1: Standard Curves
• Chemokine (C-
C motif) ligand
2
– F:300nM &
R:100nM
– 2-fold serial
dilution
Universal RNA
– SYBR Green
Fluorescence - linear scale
 Expt1: Standard Curves
• Log scale graph

• Ct value during
exponential
stage

• Ct value above
background
Expt1: Dissociation Curves
Ccl2:
single product

Ddc:
Primer dimer
 Expt1: Standard Curves
• Reproducibilit
y: R2 0.997

• Efficiency:
Slope = -
3.338 99.3%
 Expt 2: Sample Curves

Actin – 1ul cDNA

Ccl2 – 1ul cDNA

Green = nSOD1, Red = vector
Grey = G37R mutant SOD1
Yellow = G93A mutant SOD1
 Q-PCR: Analysis
ACTIN CCL2
Well Type Replicate Ct Av Ct SD Ct Av Ct SD Delta Ct dCt SD ddCt rel/con
pCEP2.3 1 18.33 25
pCEP2.3 1 18.84 25
pCEP2.3 1 18.98 18.72 0.34 24.99 25.00 0.01 6.28 0.34 0.00 1.00
pCN.5 2 18.56 21.54
pCN.5 2 18.48 21.72
pCN.5 2 18.34 18.46 0.11 21.19 21.48 0.27 3.02 0.29 -3.26 9.56
pC37.9 3 19.69 29.77
pC37.9 3 18.69 29.81
pC37.9 3 20.34 19.57 0.83 30.12 29.90 0.19 10.33 0.85 4.05 0.06
pC93A.2 4 18.72 31
pC93A.2 4 18.86 31.32
pC93A.2 4 18.7 18.76 0.09 31.04 31.12 0.17 12.36 0.19 6.08 0.01

• Use actin as internal gene control

• Express GOI relative to actin to normalise
data
• Express normalised expression of GOI in
experimental samples relative to control
 Q-PCR Results
10

1
pCEP pCN pC37 pC93

0.1

0.01

Normal SOD1 Mutant SOD1

increases decreases
expression expression of
CCL2 CCL2
 Learning objectives:
• Introduction to microarrays
• Types of arrays available
• Advantages and disadvantages
• Affymetrix GeneChips
• Manufacture processes
• Features of the GeneChip
• Experimental protocol
• Quantitative PCR
• Principles
• Detecting PCR products
• Experimental protocol
 References
• Arrays
• Nature Genetics 32 supplement (2002)
• Nature 415:964 (2002)
• www.affymetrix.com

• Q-PCR
• www.stratagene.com
• www.gene-quantification.de/chemistry