microarrays (gene chips) pioneered by pat brown in mid 1990’s to monitor thousands of mrnas...
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Microarrays (Gene Chips)
• Pioneered by Pat Brown in mid 1990’s
• To monitor thousands of mRNAs simultaneously
• Comparative Northern blot on thousands of genes
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Two mRNA samplesa ‘reference’ and a ‘experimental’
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Two mRNA samplesa ‘reference’ and a ‘experimental’
For example,
yeast cells growing via anaerobic metabolismvs.
yeast cells growing via aerobic metabolism
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Purify mRNAs from each sampleBind to –TTTTTTTTTT–beads
note that you get a population of mRNAs
Reverse Transcribe to get a population of cDNAs
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“Label” the two mRNA samples differently
Reference sample is covalently attached to the fluor Cy3 glows green under UV light
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“Label” the two mRNA samples differently
Reference sample is covalently attached to the fluor Cy3 glows green under UV light
Experimental sample is covalently attached to the fluor Cy5 glows red under UV light
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Prepare the Microarray
PCR product corresponding to every yeast gene (~6200)
Attach to a glass slide at a known location
Entire array is 18mm by 18mm (postage stamp)
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Prepare the Microarray
PCR product corresponding to every yeast gene (~6200)
Attach to a glass slide at a known location
Entire array is 18mm by 18mm (postage stamp)
PFK1
NCC1
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Mix reference and experimental cDNAs
Add to microarray
Using a microscope and a computer,
Quantitate ‘greeness’ and ‘redness’ of each spot
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DeRisi et al., Science 278: 685 1997
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Consider the NCC Gene
if NCC mRNA is equally abundant in reference and experimental population:
NCC sequenceSpot onMicroarray
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Consider the NCC Gene
if NCC mRNA is equally abundant in reference and experimental population:
Spot onMicroarray
Equal green and red=
Yellow
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Consider the NCC Gene
if NCC mRNA is more abundant in reference than experimental population (repressed by aerobic growth):
Spot onMicroarray
Spot glows green
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Consider the NCC Gene
if NCC mRNA is less abundant in reference than experimental population (induced by aerobic growth):
Spot onMicroarray
Spot glows red
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Microarrays:
• Compare amounts of several thousand mRNAs
• Red, Green, Yellow or Black
• Between two conditions, experimental and referencelooking for induction or repression
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Following the timecourse:
These seven genes all encode proteins in the electron transport chain.
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Following the timecourse:
These seven genes all encode proteins in the electron transport chain.
These sevengenes all contain the same enhancer sequence: CCAAT
Binds the transcription factor Hap2/3/4p