microarray analysis allows detection/ comparison of expression levels of multiple genes. what...
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Microarray Analysis
Allows detection/ comparison of expression levels of multiple genes.
What molecule could be used as an indicator of expression?
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DNA?
No! There are exactly two copies of every* gene in each cell.
RNA?
Perfect! The number of mRNA transcripts in a cell is proportional to expression level of the corresponding gene.
Well, almost perfect . . .
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mRNA is much less stable than DNA
Solution:
•Isolate mRNA from tissue•Immediately reverse transcribe •Incorporate a fluorescent tag
Result= Fluorescently labeled cDNA
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5’ 3’AAAAAA
TTTTTTTT Cy5Fluorescent Tag
5’ 3’AAAAAATTTTTTT
Isolate RNA from Source A (e.g., yeast cells grown under aerobic conditions)
Isolate RNA from Source B (e.g., yeast cells grown under anaerobic conditions)
Synthesize cDNAs against the mRNA templatesusing fluorescently labeled oligo dT primers.
Cy3Fluorescent Tag
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Combine the two populations of
cDNAs
Apply to microarray
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Oligonucleotide Synthesis
(How arrays are “spotted”)
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Aerobic Respiration
Anaerobic Respiration
Gene A expressed?
yes yes
Gene B expressed?
yes yes
Gene C expressed?
yes no
Gene D expressed?
no yes
Gene E expressed?
yes yes
Compare expression profiles of
yeast grown under
anaerobic conditions and
yeast grown under aerobic
conditions.
C and D
Which gene(s) should be investigated for a potential role in anaerobic respiration?
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Gene Result (Color)
C4BPA
ODC1
SIAT9
FGG
HBG1
CYP24
Our Microarray simulates expression levels of the six genes assigned for the Genome Scavenger Hunt
Red indicates expression in malignant lung tissue
Blue indicates expression in normal lung tissue
Color intensity is proportional to expression level.