Microarrays
A snapshot that captures the activity
pattern of thousands of genes at once.
Custom spotted arrays
Affymetrix GeneChip
Gene Expression: Transcription
The majority of genes are expressed as the proteins they
encode. This process occurs in two steps:
Transcription: DNA
RNA
Translation: RNA
Protein
By measuring amounts of mRNA for any specific gene, we
can identify changes in gene regulation.
Genes which behave very differently in a disease state
versus a normal state can be inferred to have something to
do with a disease.
Diagnosis
???
???
Sample Collection
Integrity of sample is
CRITICAL!!!
Immediately snap freeze
in liquid nitrogen
Tissue characteristics that make
RNA isolation challenging….
Tissue
Characteristics
Symptoms
Representative
tissue types
Fibrous tissues
Difficult to
homogenize
Heart muscle
Skeletal Muscle
Nucleic acid-rich
Viscous lysate,
incomplete phase
separation
Spleen
Thymus
Protein- and lipid-rich
White ppt material
in aqueous phase of
extraction
Brain
Nuclease-rich
Degraded RNA
Spleen
Pancrease
Thymus
Total RNA isolation methods
RNeasy Stabilization and
Total RNA Isolation System
Yields of Total RNA isolated from Tissue
Maximum
Amount to
Process
Average
Yield per
Prep (mg)
30mg
20mg
133
46
Average
Yield per mg
Tissue (mg)
4.4
2.3
Heart
Spleen
Brain
Lung
60mg
15mg
60mg
60mg
16
79
39
36
0.3
5.3
0.7
0.6
Muscle
Breast
30mg
70mg
22
63
0.7
0.9
Samples
Liver
Kidney
Challenges in samples size
Core biopsies
Fine needle aspirate
Laser capture microdissection
RNA amplification
Maintain integrity of sample
Maintain quantitative relationships
Maintain reproducibility
Agilent 2100 Bioanalyzer
RNA Quality
RNA quality
Ribosomal products are very abundant and provide
an easy assessment of the RNA integrity of a sample.
Good rRNA ratio = >1.7
Fair rRNA ratio = 1.2-1.6
Bad rRNA ratio= <1.2
Microarray Platforms
•Oligonucleotide-based arrays
•25mers spotted on a glass wafer, Affymetrix
GeneChip arrays
•Custom spotted 50-80mers generated from
known sequences.
•cDNA
•Inserts from cDNA libraries
•PCR products generated from gene specific or
universal primers
Array Fabrication
Affymetrix GeneChip arrays
Custom spotted arrays
GeneChip Probe Array
®
GeneChip Probe Arrays
®
GeneChip Probe Array
Hybridized Probe Cell
Single stranded, fluorescently
labeled DNA target
Oligonucleotide probe
1.28cm
*
*
*
*
*
24µm
Each probe cell or feature contains
millions of copies of a specific
oligonucleotide probe
Over 250,000 different probes
complementary to genetic
information of interest
Image of Hybridized Probe Array
Synthesis of Ordered
Oligonucleotide Arrays
Light
(deprotection)
Mask
OOOOO
TTOOO
HO HO O O O
T–
Substrate
Light
(deprotection)
Mask
CATAT
AGCTG
TTCCG
TTCCO
TTOOO
C–
Substrate
REPEAT
Probe Tiling Strategy
Gene Expression
(25-mer)
Affymetrix Experimental Design
(one sample per array)
Biotin - labeled
cRNA transcript
Cells
B
+
Poly (A)
RNA
Or
Total RNA
IVT
cDNA
(10mg)
Biotin-UTP
Biotin-CTP
B
B
B
B
B
B
Fragment
heat, Mg2+
B
Hybridize
B
B
B
B
Wash & Stain
Scan
(8 minutes)
(75 minutes)
(16 hours)
Biotin - labeled cRNA
fragments
Add Oligo B2 &
Staggered Spike
Controls
Custom Spotted
Array Fabrication
cDNA
Oligonucleotides (50-80mers)
Poly-l-lysine slides or
commercially available slides
Primary amine groups (NH3+) attached covalently to the glass surface (rectangles). The amines carry a
positive charge at neutral pH, allowing attachment of native DNA (red ribbons) through the formation of
ionic bonds with the negatively charged phosphate backbone (middle panel). Electrostatic attachment is
supplemented by treatment with ultraviolet light or heat, which induces covalent attachment of the DNA
to the surface (right panel). The combination of electrostatic binding and covalent attachment couples the
DNA to the substrate is a highly stable manner.
Printing with TeleChem printhead
and 16 pins (SMP 3,90-100 microns)
GeneMachines OmniGrid Arrayer
With Robiotic Arm
TeleChem Micro Spotting pins and printhead
48 pin printhead
SMP3 pin
Prints 90-100 mm spots
Operon Oligonucleotide
Genome Sets
Human
21,656
Mouse
16,463
Rat
6,273
Yeast
6,307
C. albicans 6,266
cDNA Arrays
Ferret
Songbird
Mouse full length
Cryptococcus
L. Pine
Spotted Microarray Experimental Design
(2 samples per array, i.e.tumor vs. reference)
ratio > 1.0
ratio = 1.0
ratio < 1.0
POPO-3 stain
•Detect depletion of sample due to vaporation.
•Detect spots of low concentration of DNA.
POPO-3 is a intercalating cyanine dimer, which excites and
detects the ssDNA at the Cy3 channel of a microarray
scanner (532 wavelength).
Data Collection
and Analysis
Axon GenePix 4000B
Affymetrix GeneChip System
.DAT file and .CEL file
Absolute Analysis
Treated vs. reference RNA
Upregulated or overexpressed, ratio > 1.0
Equal expression, ratio = 1.0
Downregulated or underexpressed, ratio < 1.0
GenePix Pro Features
•Feature Viewer
P = pixel intensity
F = feature intensity
B = background intensity
Rp = ratio of pixel intensities
Rm = ratio of means
mR = median of ratios
rR = regression ratio
• Results
GenePix Pro Features
*Ratio of median Cy5/Cy3
Challenges in obtaining reliable
Microarray Data
Biological Variation
Strains
Animals
Tissues
Time points
Processing variation caused by
Quality of experimental sample
Labeling effects
Hybridization effects
Background effects
Challenges in obtaining reliable
Microarray Data
How many ???
More replication is needed for…
Finding small differences in genes expressed at modest levels
Experiments using tissue samples
Experiments with NO confirmatory testing (i.e. RT-PCR)
Less replication is needed for…
Finding gross patterns among highly expressed genes
Experiments using cell line samples
Experiments incorporating confirmatory testing such as
Northern blots or Real-time PCR
Some issues to consider before designing
microarray experiments
Scientific
Aims of the experiment
Specific questions and priorities between them.
How will the experiments answer the questions posed?
Practical (Logistic)
Types of mRNA samples: reference, control, treatment, mutant, etc
Amount of material needed for entire experiment
The number of slides available for the experiment.
Other Information
The experimental process prior to hybridization: sample isolation, mRNA
extraction, amplification, labelling,…
Controls planned: positive, negative, ratio, etc.
Verification method: Northern, RT-PCR, in situ hybridization, etc.
Improving your Experiment Through Replication
Biological Variation
Strains
Animals
Tissues
Time points
Processing variation caused by
Quality of experimental sample
Labeling effects
Hybridization effects
Background effects
More replication is needed for…
Finding small differences in genes expressed at modest levels
Experiments using tissue samples
Experiments with NO confirmatory testing (i.e. RT-PCR)
Less replication is needed for…
Finding gross patterns among highly expressed genes
Experiments using cell line samples
Experiments incorporating confirmatory testing such as
Northern blots or Real-time PCR
$$$$BUDGET…
Affymetrix array assay
$850.00 /sample
Spotted Microarray assay
$150.00/sample
(450.00/array, 400.00/reagents and labor)
(75.00/array, 75.00/reagents and labor)