Uses of Microarrays in Research

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Uses of Microarrays in Research

• Anne Rosenwald
• Biology Department
• Georgetown University

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Microarrays in ResearchA Survey of PubMed
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Recent Microarray Papers I. New
Techniques/Applications

• 5,000 RNAi experiments on a chip
• Lehner and Fraser (2004) Nat Methods 1, 103
• RNA living-cell microarrays for loss-of-function
  screens in Drosophila melanogaster cells
• Wheeler et al. (2004) Nat Methods 1, 127
• Spots on chip contain dsRNA
• Chip incubated with Drosophila cells
• Cells induced to take-up RNA
• Are cells alive or dead?
• Do cells have phosphorylated Akt?
• Do cells have altered actin fibrils?

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Recent Microarray Papers I. New
Techniques/Applications

• Transcriptional regulatory networks in
  Saccharomyces cerevisiae
• Lee et al. (2002) Science 298 799-804
• ChIP-on-chip

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Recent Microarray Papers II. Improved Methods
for Analysis/Access

• Reproducibility and statistical rigor
• outbred organisms (i.e. humans)
• do different platforms give the same answers?
• Tools for analysis
• Tools for access and annotation
• an example based on Affymetrix chips
• GeneCruiser a web service for the annotation of
  microarray data Liefeld et al. Bioinformatics
  (2005) Jul 19 epub
• can incorporate GO terms and link info with
  SwissProt, RefSeq, LocusLink, etc.
• Primarily for mouse and human data

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Recent Microarray Papers III. Scientific
Endeavors

• Mutational change compare wild type to mutant
• Tissue-specific gene expression
• Environmental change compare same organism in
  two different environments
• Development compare different stages along a
  particular lineage
• Therapeutics compare in cells/tissues treated
  with and without the drug of interest
• Investigate changes in gene copy number
• Cancer compare tumor with normal surrounding
  tissue
• 2005 papers with term microarray 2450
• Of those, also with term cancer 624 (25)

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Recent Microarray Papers III. New Scientific
Endeavors

• Transgenic C. elegans as a model in Alzheimer's
  research
• Curr Alzheimer Res. 2005 Jan2(1)37-45.
• Compared wild type worms with worms expressing
  human Ab
• Behavior and the limits of genomic plasticity
  power and replicability in microarray analysis of
  honeybee brains
• Genes Brain Behav. 2005 Jun4(4)267-71
• Compared bees with long-standing behavioral
  differences (nursers v. foragers)
• Compared recently hatched bees beginning to
  express behavioral differences (nursers v.
  foragers v. gravetenders)

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Some basic yeast biology

• Yeast come in two mating types
• MATa
• MATa
• Can live either as haploids or as diploids
• diploids referred to as MATa/a
• Haploids of opposite mating type can mate to form
  new diploids
• Diploids can be induced to undergo meiosis
  (sporulation) to make new haploids

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Yeast resources

• General website for Saccharomyces (SGD)
• http//www.yeastgenome.org/
• Materials available
• 5500 genes cloned with tags for purification
• TAP-tagged fusion collections
• HA-tagged fusion collections
• GFP-tagged fusion collections
• Insertional mutant collections
• Knockout collections
• Most of these available from OpenBiosystems
• www.openbiosystems.com

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The yeast knockout collection

• Yeast knockout resources
• MATa/a heterozygous diploids (entire genome)
• MATa haploids (non-essentials)
• MATa haploids (non-essentials)
• MATa/a homozygous diploids (non-essentials)
• Yeast knockout website
• http//www-sequence.stanford.edu/group/yeast_delet
  ion_project/deletions3.html

I have this collection, so if theres a mutant
you want, let me know.
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The yeast knockout collection
http//www-sequence.stanford.edu/group/yeast_delet
ion_project/deletions3.html
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Using the knockouts for microarrays

• A Robust Toolkit for Functional Profiling of the
  Yeast Genome
• Pan et al. (2004) Mol Cell 16, 487
• Takes advantage of the MATa/a heterozygous
  diploid collection
• identifies synthetic lethal interactions via
  diploid-based synthetic lethality analysis by
  microarrays (dSLAM)
• Uses dSLAM to identify those strains that upon
  knockout of a query gene, show growth defects
• synthetic lethal (the new double mutant dead)
• synthetic fitness (the new double mutant slow
  growth)

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Step 1 Creating the haploid convertible
heterozygotes
Important point This HIS3 gene is only expressed
in MATa haploids, not in MATa haploids or MATa/a
diploids So in other words, can select against
MATa/a diploids to ensure youre looking at only
haploids later on.
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Step 2 Inserting the query mutation
Knockout one copy of your gene of interest (Your
Favorite Gene) with URA3
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Step 3 Make new haploids and select for strains
of interest
Sporulate to get new haploids
Select on his medium to ensure only haploids
survive (no diploids)
selects against query mutation so genotype is
xxxDKanMX YFG1
selects for query mutation so genotype is
xxxDKanMX yfg1URA3
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Reminder about YKO construction
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Step 4 Prepare genomic DNA and do PCR with
common TAG sequences
U1
D1
U2
D2
Using common oligos U1 and U2 (or D1 and D2)
amplifies the UPTAG (or DNTAG) sequence unique to
each of the KOs
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Step 4 Prepare genomic DNA and do PCR with
common TAG sequences
The two different conditions are labeled with two
different colors
The labeled DNA is then incubated with a TAG
microarray
The PCR reactions create a mixture of TAGs
(representing all the strains in the pool), since
each KO has a unique set of identifier tags
(UPTAG and DNTAG) bounded by common
oligonucleotides
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Evidence this really works part I
On average, the intensity is the same before and
after 1 copy of the CAN1 gene is knocked out
Strains
x-axis
y-axis
XXX/xxxDKanMX CAN1/CAN1
XXX/xxxDKanMX CAN1/can1DMFA1pr-HIS3
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Evidence this really works part II
Red spots illustrate that fraction of the strains
with KOs in essential genes, so when haploid, not
present in pool
Strains
x-axis
y-axis
DIPLOIDS XXX/xxxDKanMX CAN1/can1DMFA1pr-HIS3
HAPLOIDS XXX or xxxDKanMX can1DMFA1pr-HIS3
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Another variation Drug sensitivity
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Another variation Drug sensitivity
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Summary

• If you can compare two different conditions and
  you have a way to stick things to slides, some
  sort of microarray is possible!