1. Affymetrix vs. glass slide based arrays
Short oligonucleotides
Many oligos per gene
Single sample hybridized to chip
Fixed platform.
Not universally available for each species.
EXPENSIVE!
Glass slide
Long oligonucleotides or PCR products
A single oligo or PCR product per gene
Two samples hybridized to chip
Flexible platform
LESS EXPENSIVE especially for small microbial genomes

2. Affymetrix Array With 8,000 Genes
16 perfect match oligos
16 mismatch oligos
Aharoni and Vorst, in press

3. Spotted Microarrays DNA representing gene spotted on slide
Direct comparison between two fluorescent labeled RNA samples

4. Competitive hybridization is the key to two-color DNA microarrays!
Competitive hybridization - both samples have the same opportunity to hybridize.

5. PCR vs oligo arrays Long oligonucleotides Single stranded
PCR (cDNA)
Double stranded
Less specificity
Significant cost and time involved in sample preparation.
PCR products less stable?
Increased signal strength
Intensity doesn’t correlate with expression levels. Products all different sizes.
Long oligonucleotides
Single stranded
More specificity
Synthesized by company at significant cost.
Oligos stable over time.
Less signal strength
Intensity does correlate with expression levels. All oligos similar Tms.

6. Designing oligonucleotides for DNA microarrays
Free software available - OligoArray
Oligo characteristics
Length - governs specificity and sensitivity
No secondary structure
Fixed GC range
Melting temperature
Cost ~ $10 per oligo
Which strand do you use?

7. Printing DNA microarrays
Focus on glass slide based arrays
Direct printing of pre-synthesized oligos on a glass surface.
Use robotics for precision printing.
Many slide chemistries used.
Building of oligonucleotides on a glass surface
Ink-jet methodology, micro mirrors.

8. Labeling RNA or DNA with Cy3 or Cy5.
Cy3 and Cy5 - most often used fluorescent molecules used to label samples for microarray analysis.
Absorb light at one wavelength and emit at another.
Emission and Excitation spectra do not overlap significantly.
In arrays Cy3 and Cy5 are usually false colored green (Cy3) and red (Cy5) for ease of visualization.

9. More labeling
Direct incorporation - incorporates Cy3-or Cy5-dNTP directly into cDNA
RNA to cDNA - reverse transcriptase
DNA to DNA - DNA polymerase
Big problem - Cy3 and Cy5 are not incorporated with same efficiency.
Indirect incorporation - preferred method.
Incorporate an aminoallyl-dUTP molecule during reverse transcription of RNA to cDNA.
Chemically couple Cy3 or Cy5 dye after cDNA is made.
Coupling is efficient with both dyes.

10. Image Analysis GenePix (Axon) Quantitate genes Normalize data
16 bit image

11. Data from DNA microarray experiments
Expressed as relative expression levels
Cy5/Cy3
Not absolute expression
Data must be normalized
Data is log2 transformed in most cases
Excel spreadsheets - ~30 columns and 4300 rows of data per experiment.
Data storage and analysis issues.

12. What is a significant change in gene expression?
2 fold? 5 fold?
Statistical representation of the data
Significance analysis for microarrays
Standard t-tests
Iterative outlier analysis
Biological replicates vs. technical replicates
Biological replicates are essential for generating significant data.