microarrays (gene chips) pioneered by pat brown in mid 1990’s to monitor thousands of mrnas...

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 samplesa ‘reference’ and a ‘experimental’

Two mRNA samplesa ‘reference’ and a ‘experimental’

For example,

yeast cells growing via anaerobic metabolismvs.

yeast cells growing via aerobic metabolism

Purify mRNAs from each sampleBind 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 sequenceSpot onMicroarray

Consider the NCC Gene

if NCC mRNA is equally abundant in reference and experimental population:

Spot onMicroarray

Equal green and red=

Yellow

Consider the NCC Gene

if NCC mRNA is more abundant in reference than experimental population (repressed by aerobic growth):

Spot onMicroarray

Spot glows green

Consider the NCC Gene

if NCC mRNA is less abundant in reference than experimental population (induced by aerobic growth):

Spot onMicroarray

Spot glows red

Microarrays:

• Compare amounts of several thousand mRNAs

• Red, Green, Yellow or Black

• Between two conditions, experimental and referencelooking 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 sevengenes all contain the same enhancer sequence: CCAAT

Binds the transcription factor Hap2/3/4p