1. Introduction to Microarray
Dr G. P. S. Raghava

2. Molecular Biology Overview
Nucleus
Cell
Chromosome
Gene (DNA)
Protein
Gene (mRNA),
single strand

3. Measuring Gene Expression
Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell. Measuring protein would be more direct, but is currently harder.

4. (RT)

5. The Goals Basic Understanding
Arrays can take a snap shot of which subset of genes in a cell is actively making proteins
Heat shock experiments
Medical diagnosis
Microarrays can indicate where mutations lie that might be linked to a disease. Still others are used to determine if a person’s genetic profile would make him or her more or less susceptible to drug side effects
1999 – A genechip containing 6800 human genes was used distinguish between myeloid leukemia and lympholastic leukemia using a set of 50 genes that have different activity levels
Drug design
Pharmaceutical firms are in a rush to translate the human genome results into new products
Potential profits are huge
First, though, they must figure out what the genes do, how they interact, and how they relate to diseases.
Evaluation, Specificity, Response

6. Microarray Potential Applications
Biological discovery
new and better molecular diagnostics
new molecular targets for therapy
finding and refining biological pathways
Recent examples
molecular diagnosis of leukemia, breast cancer, ...
appropriate treatment for genetic signature
potential new drug targets

7. History 1980s: antibody-based assay (protein chip?)
~1991: high-density DNA-synthetic chemistry (Affymetrix/oligo chips)
~1995: microspotting (Stanford Univ/cDNA chips)
replacing porous surface with solid surface
replacing radioactive label with fluorescent label
improvement on sensitivity

8. What is a DNA Microarray?
genes or gene fragments attached to a substrate (glass)
Tens of thousands of spots/genes
=entire genome in 1 experiment
A Revolution in Biology
Hybridized slide
Two dyes
Image analyzed

9. Gene Expression Microarrays
The main types of gene expression microarrays:
Short oligonucleotide arrays (Affymetrix);
cDNA or spotted arrays (Brown/Botstein).
Long oligonucleotide arrays (Agilent Inkjet);

10. Terms/Jargons Stanford/cDNA chip one slide/experiment one spot
1 gene => one spot or few spots(replica)
control: control spots
control: two fluorescent dyes (Cy3/Cy5)
Affymetrix/oligo chip
one chip/experiment
one probe/feature/cell
1 gene => many probes (20~25 mers)
control: match and mismatch cells.

11. 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

12. DNA Microarrays
Each probe consists of thousands of strands of identical oglionucleotides
The DNA sequences at each probe represent important genes (or parts of genes)
Printing Systems
Ex: HP, Corning Inc.
Printing systems can build lengths of DNA up to 60 nucleotides long
1.28 x cm glass wafer
Each “print head” has a ~100 m diameter and are separated by ~100 m. ( 5,000 – 20,000 probes)
Photolithographic Chips
Ex: Affymetix
1.28 x 1.28 cm glass/silicon wafer
24 x 24 m probe site ( 500,000 probes)
Lengths of DNA up to 25 nucleotides long
Requires a new set of masks for each new array type
GeneChip

13. 10% Biotin-labeled Uracil
The Process
Cells
Poly-A
RNA
AAAA
cDNA L
IVT
10% Biotin-labeled Uracil
Antisense cRNA
Fragment (heat, Mg2+)
Labeled
fragments
Hybridize
Wash/stain
Scan
(In-vitro
Transcription)

14. Hybridization and Staining
GeneChip
Biotin
Labeled cRNA
Hybridized Array L L L L + L L L + L L L L L
SAPE
Streptavidin-
phycoerythrin

15. Microarray Data First, the Problems:
The fabrication process is not error free
Probes have a maximum length nucleotides
Biologic processes such as hybridization are stochastic
Background light may skew the fluorescence
How do we decide if/how strongly a particular gene is being expressed?
Solutions to these problems are still in their infancy

16. Affymetrix “Gene chip” system
Uses 25 base oligos synthesized in place on a chip (20 pairs of oligos for each gene)
RNA labeled and scanned in a single “color”
one sample per chip
Can have as many as 20,000 genes on a chip
Arrays get smaller every year (more genes)
Chips are expensive
Proprietary system: “black box” software, can only use their chips

17. cDNA Microarray Technologies
Spot cloned cDNAs onto a glass microscope slide
usually PCR amplified segments of plasmids
Label 2 RNA samples with 2 different colors of flourescent dye - control vs. experimental
Mix two labeled RNAs and hybridize to the chip
Make two scans - one for each color
Combine the images to calculate ratios of amounts of each RNA that bind to each spot

18. cDNA microarrays
Compare the genetic expression in two samples of cells
PRINT
cDNA from one gene on each spot
SAMPLES
cDNA labelled red/green
e.g. treatment / control
normal / tumor tissue

19. HYBRIDIZE
Add equal amounts of labelled cDNA samples to microarray.
SCAN
Laser
Detector

20. “Long Oligos”
Like cDNAs, but instead of using a cloned gene, design a base probe to represent each gene
Relies on genome sequence database and bioinformatics
Reduces cross hybridization
Cheaper and possibly more sensitive than Affy. system

21. Images from scanner Resolution
standard 10m [currently, max 5m]
100m spot on chip = 10 pixels in diameter
Image format
TIFF (tagged image file format) 16 bit (65’536 levels of grey)
1cm x 1cm image at 16 bit = 2Mb (uncompressed)
other formats exist e.g.. SCN (used at Stanford University)
Separate image for each fluorescent sample
channel 1, channel 2, etc.

22. Processing of images Addressing or gridding Segmentation
Assigning coordinates to each of the spots
Segmentation
Classification of pixels either as foreground or as background
Intensity determination for each spot
Foreground fluorescence intensity pairs (R, G)
Background intensities
Quality measures

23. Images in analysis software
The two 16-bit images (Cy3, Cy5) are compressed into 8-bit images
Display fluorescence intensities for both wavelengths using a 24-bit RGB overlay image
RGB image :
Blue values (B) are set to 0
Red values (R) are used for Cy5 intensities
Green values (G) are used for Cy3 intensities
Qualitative representation of results

24. Images : examples Pseudo-colour overlay Cy3 Cy5 Spot colour
Signal strength
Gene expression
yellow
Control = perturbed
unchanged
red
Control < perturbed
induced
green
Control > perturbed
repressed

25. Quantification of expression
For each spot on the slide we calculate
Red intensity = Rfg - Rbg
(fg = foreground, bg = background) and
Green intensity = Gfg - Gbg
and combine them in the log (base 2) ratio
Log2( Red intensity / Green intensity)

26. Gene Expression Data = slide 1 slide 2 slide 3 slide 4 slide 5 …
On p genes for n slides: p is O(10,000), n is O(10-100), but growing,
Slides
slide 1 slide 2 slide 3 slide 4 slide 5 …
Genes 3
Gene expression level of gene 5 in slide 4 =
Log2( Red intensity / Green intensity)
These values are conventionally displayed
on a red (>0) yellow (0) green (<0) scale.

27. 16-bit TIFF files (Rfg, Rbg), (Gfg, Gbg) R, G 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

28. Quality control (-> Flag)
How good are foreground and background measurements ?
Variability measures in pixel values within each spot mask
Spot size
Circularity measure
Relative signal to background intensity
Dapple:
b-value : fraction of background intensities less than the median foreground intensity
p-score : extend to which the position of a spot deviates from a rigid rectangular grid
Flag spots based on these criteria

29. Replication Why? What is it? To reduce variability
To increase generalizability
What is it?
Duplicate spots
Duplicate slides
Technical replicates
Biological replicates

30. Practical Application of DNA Microarrays
DNA Microarrays are used to study gene activity (expression)
What proteins are being actively produced by a group of cells?
“Which genes are being expressed?”
How?
When a cell is making a protein, it translates the genes (made of DNA) which code for the protein into RNA used in its production
The RNA present in a cell can be extracted
If a gene has been expressed in a cell
RNA will bind to “a copy of itself” on the array
RNA with no complementary site will wash off the array
The RNA can be “tagged” with a fluorescent dye to determine its presence
DNA microarrays provide a high throughput technique for quantifying the presence of specific RNA sequences

31. Analysis and Management of Microarray Data
Magnitude of Data
Experiments
genes in human
320 cell types
2000 compunds
3 times points
2 concentrations
2 replicates
Data Volume
4*1011 data-points
1015 = 1 petaB of Data

32. Thanks