Transcript Lecture_7_2005

Affymetrix vs. glass slide based
arrays
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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
8,000 Genes
16 perfect
match oligos
16 mismatch
oligos
Aharoni and Vorst, in press
Spotted 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
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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
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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.