Affymetrix vs. glass slide based arrays

1
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

2
Affymetrix Array With 8,000 Genes
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

• PCR (cDNA)
• Double stranded
• Less specificity
• Significant cost and time involved in sample
  preparation.
• PCR products less stable?
• Increased signal strength
• Intensity doesnt 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.