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Microarrays
A snapshot that captures the activity pattern of thousands of genes at once.
Custom spotted arrays Affymetrix GeneChip
Microarray Process
Practical Applications of Microarrays
Gene Target DiscoveryBy allowing scientists to compare diseased cells with normal cells, arrays can be used to discover sets of genes that play key roles in diseases. Genes that are either overexpressed or underexpressed in the diseased cells often present excellent targets for therapeutic drugs.
Pharmacology and ToxicologyArrays can provide a highly sensitive indicator of a drug’s activity(pharmacology) and toxicity (toxicology) in cell culture or test animals. This information can then be used to screen or optimize drug candidates prior to launching costly clinical trials.
DiagnosticsArray technology can be used to diagnose clinical conditions by detectinggene expression patterns associated with disease states in either biopsysamples or peripheral blood cells.
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
GeneChip® Instrument System
FluidicsStation
Scanner made byHewlett-Packard
Computer Workstation
GeneChip® Probe Array
GeneChip® Probe Arrays
24µm
Each probe cell or feature containsmillions of copies of a specificoligonucleotide probe
Image of Hybridized Probe Array
Over 250,000 different probes complementary to geneticinformation of interest
Single stranded, fluorescentlylabeled DNA target
Oligonucleotide probe
* **
**
1.28cm
GeneChip Probe Array Hybridized Probe Cell
Synthesis of Ordered Oligonucleotide Arrays
O O O O O
Light(deprotection)
HO HO O O O T T O O O
T T C C O
Light(deprotection)
T T O O O
C A T A T
A G C T GT T C C G
Mask
Substrate
Mask
Substrate
T –
C –REPEAT
Probe Tiling StrategyGene Expression
(25-mer)
Gene ExpressionGene ExpressionTiling StrategyTiling Strategy
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[ ][ ]
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Uninduced Induced
80 separate hybridization events are involved determining differential gene expression of a transcript between two samples
40 separate hybridization events are involved in determining the presence or absence of a transcript
Starting material for Microarrays
PlatformAffymetrix
Poly (A)+ mRNA ~2 gTotal RNA ~10 g
Spotted arraysPoly (A) + ~0.4 – 2 gTotal RNA 10 -100 g
Experimental Design
cDNA Fragmentheat, Mg2+
Wash & Stain
Scan
Hybridize
(16 hours)
Biotin - labeled cRNA transcript
Poly (A)+
RNAOr
Total RNA
IVT
Biotin-UTPBiotin-CTP
Biotin - labeled cRNA fragments
Cells
B B B BB B
B
B
B
B
B
B
(8 minutes)
(75 minutes)
Add Oligo B2 & Staggered Spike
Controls
Normalization and Scaling
Non-biological factors can contribute to the variability of datain many biological assays, therefore it is important to minimizethe non-biological differences. Factors that may contribute to variation include:
•Amount and quality of target hybridized to array•Amount of stain applied•Experimental variables
The data can be normalized from:•a limited group of probe sets •all probe sets
Thus the normalization of the array is multiplied by a Normalization Factor (NF) to make its Average Intensityequivalent to the Average Intensity of the baseline array.
Average intensity of an array is calculated by averaging allthe Average Difference values of every probe set on the array, excluding the highest 2% and lowest 2% of the values.
Normalization and Scaling
Data Processing:•Analytical Spreadsheet can Handle Millions of Rows or Columns•Scaling & Normalization (e.g. standardize, log-scale, log & linear scale, power)•Sort rows by Value or by Similarity to Prototype (find genes most similar to specified prototype)•Missing Data Handling (e.g. analysis, casewise deletion, imputation)
Cluster and Tree View
Microarray Process
Easy-To-Spot™ Products (Incyte Genomics)
•Every clone is sequence-verified prior to PCR • PCR products are purified to remove excess salts, unincorporated nucleotides, primers, and particulates • Quality controlled production process with failure rate1 of less than 10%
• 8,734 PCR products from sequenced-verified clones from the UniGene database from NCBI, average length is greater than 500 nucleotides•Between 1-3 ug of DNA per well. Enough to fabricate 500 to 1,000 arrays • Corresponding clones available for purchase for further research
Products used for spotting
ClontechClontechAtlas™ Glass Fluorescent Labeling KitAtlas™ Glass Fluorescent Labeling Kit
Indirect labeling
StratageneFairPlay™ Microarray Labeling Kit
Simple, highly sensitive technique requires less starting RNA, and creates evenly labeled DNA without dye bias.
•Uniform incorporation of fluorescent dyes produces more reliable signals
•High sensitivity to detect low-copy signals
•Requires only 10 to 20 µg of total RNA or 0.4 to 1 µg of polyA RNA
Complete Yeast Genome Oligo Set• Optimized 70-mer oligonucleotides for each of the 6,307 open reading frames (ORFs) of Saccharomyces cerevisiae from the Saccharomyces Genome Database (SGD) at Stanford University
•The amount of sample provided with each set is sufficient to print between 2000 and 6000 slides, depending on the printing procedure used.
Human Genome Oligo Set •This Array-Ready Oligo Set™ contains arrayable 70-mers representing 13,971 well-characterized human genes from the UniGene database. This database is located at the National Center for Biotechnology Information.
•All 70-mer oligonucleotides in the Human Genome Oligo Set were designed from the representative sequences in the UniGene database, Hs build #119. The set also contains 29 controls.
Array Ready Oligo set (Operon Technologies)
GeneMachine Omni Grid Arrayer
Printing Pin
Axon GenePix4000A Scanner
• 10mm pixel size• Simultaneously scans array slides at two wavelengths• User-selectable laser power• User-selectable focus poisitions
GenePix Pro Features
• Auto Align
Before Auto Align After Auto Align
GenePix Pro Features
•Feature Viewer
P = pixel intensityF = feature intensityB = background intensityRp = ratio of pixel intensitiesRm = ratio of meansmR = median of ratiosrR = regression ratio
GenePix Pro Features
•Feature Pixel Plot
GenePix Pro Features
•Histogram
GenePix Pro Features
•Scatter Plot
Spotted glass slide microarrays
AdvantagesLow cost per array
Custom gene selectionAny species
Competitive hybridizationOpen architecture
DisadvantagesClone management
Clone costQuality control
AdvantagesStream line production
Large number of genes and ESTs/chipSeveral number of species
DisadvantagesSystem cost
GeneChip costPropietary system
Limits on customizing
Affymetrix GeneChip system
GeneCards Database
Challenges in analyzing Microarray Data
•Amount of DNA in spot is not consistant•Spot contamination•cDNA may not be proportional to that in the tissue•Low hybridization quality •Measurement errors•Spliced variants•Outliers•Data are high-dimensional “multi-variant”•Biological signal may be subtle, complex, non linear, and buried in a cloud of noise•Normalization•Comparison across multiple arrays, time points, tissues, treatments•How do you reveal biological relationships among genes?•How do you distinguish real effect from artifact?
Factors to consider in designing microarray experiments
•Need to do lots of control experiments-validate method
•Do replicate spotting, replicate chips, and reverse labelingfor custom spotted chips
•Do pilot studies before doing “mega chip” experiments
•Don’t design experiment without replication; nothing will be learned from a single failed experiment
•Design simple (one-two factor) experiments, i.e. treatment vs. untreatment
•Understand measurement errors
•In designing Databases; they are useful ONLY if quality of data is assured
•Involve statistical colleagues in the design stages of your studies
Once you have identified an interesting expression pattern, what comes next?
•With some arrays it is possible to purchase clones of interest for further experimentation.
•Confirm that the particular clone you now have in your hand shows the expression pattern so indicated by the array, quantitating individual mRNA species.
•RT-PCR, Relative, quantitative RT-PCR uses an internal standard to monitor each reaction and allow comparisons between different reactions to be made.• Competitive RT-PCR --a competition between a known amount of a template and an unknown target.
•Northern analysis
