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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
Two mRNA samples
a ‘reference’ and a ‘experimental’
Two mRNA samples
a ‘reference’ and a ‘experimental’
For example,
yeast cells growing via anaerobic metabolism
vs.
yeast cells growing via aerobic metabolism
Purify mRNAs from each sample
Bind to –TTTTTTTTTT–beads
note that you get a population of mRNAs
Reverse Transcribe to get a population of cDNAs
“Label” the two mRNA samples differently
Reference sample is covalently attached to the fluor
Cy3 glows green under UV light
“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
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)
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
Mix reference and experimental cDNAs
Add to microarray
Using a microscope and a computer,
Quantitate ‘greeness’ and ‘redness’ of each spot
DeRisi et al.,
Science
278: 685 1997
Consider the NCC Gene
if NCC mRNA is equally abundant in reference and
experimental population:
NCC sequence
Spot on
Microarray
Consider the NCC Gene
if NCC mRNA is equally abundant in reference and
experimental population:
Equal green and red
=
Yellow
Spot on
Microarray
Consider the NCC Gene
if NCC mRNA is more abundant in reference than
experimental population (repressed by aerobic growth):
Spot glows green
Spot on
Microarray
Consider the NCC Gene
if NCC mRNA is less abundant in reference than
experimental population (induced by aerobic growth):
Spot glows red
Spot on
Microarray
Microarrays:
• Compare amounts of several thousand mRNAs
• Red, Green, Yellow or Black
• Between two conditions, experimental and reference
looking for induction or repression
Following the
timecourse:
These seven
genes all encode
proteins in the
electron
transport chain.
Following the
timecourse:
These seven
genes all encode
proteins in the
electron
transport chain.
These seven
genes all contain the same enhancer sequence: CCAAT
Binds the transcription factor Hap2/3/4p