Affymetrix vs. glass slide based arrays

• Affymetrix• 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

Affymetrix Array With Affymetrix Array With 8,000 Genes8,000 Genes

16 perfect match oligos

16 mismatch oligos

Aharoni and Vorst, in press

Spotted MicroarraysSpotted Microarrays

• DNA representing gene spotted on slide

• Direct comparison between two fluorescent labeled RNA samples

Competitive hybridization is the key to two-color DNA

microarrays!• Competitive hybridization - both samples

have the same opportunity to hybridize.

PCR vs oligo arrays

• 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.

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?

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.

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.

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.

Image Analysis

• GenePix (Axon)

• Quantitate genes

• Normalize data

• 16 bit image

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.

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.