Download Gene expression An organism`s genome is the complete set of

Gene expression
An organism’s genome is the complete
set of genes in each of its cells. Given
an organism, every one of its cells has
a copy of the exact same genome, but
◆ not all its cells express the same genes
◆ different genes express under different
conditions
Measure the levels of the various mRNAs in
a cell in a specific state ⇒ gene expression
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profile of cell in that state ⇒ cell state.
DNA microarrays
One of the most promising tools
for obtaining gene expression
data is the DNA microarray.
A DNA microarray is a tiny glass
slide on which genes (purified
single-stranded cDNA sequences
in solution) have been robotically
printed in an (approximately)
rectangular array; each spot on
the array corresponds to a single
gene.
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Experimental procedure
◆ Prepare DNA microarray.
◆ Prepare labeled test sample.
cellular contents
→
mRNA
(isolate & purify)
→ cDNA
(reverse
transcription)
→
(add flourescent
dye)
sample
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Experimental procedure
(continued)
◆ Flood the microarray with the labeled
sample.
◆ Whenever there is cDNA in the array
complementary to mRNA in the sample,
the two will hybridize.
◆ Hybridization is allowed to take place,
then the array is washed and dried.
◆ The array is scanned with
a laser microscope.
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Scanned image
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Interpreting the scanned image
◆ High intensity spot ⇒ the DNA at that
spot corresponds to some RNA in sample.
◆ Low intensity spot ⇒ no RNA in sample
that corresponds to the DNA at that spot.
◆ Intensity ~ RNA abundance.
◆ For any gene, can compare intensities
across different samples (but shouldn’t
compare intensities for different genes for
the same sample).
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Comparing two scanned images
Control
Treatment
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Paradigm
[expressed] gene ➡ mRNA
amount of
mRNA in cell
➡
transcription level
of gene
change in state of cell
→ change in gene expression pattern
within cell
→ change in mRNA abundance in cell
→ change in spot intensity pattern
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Uses of DNA microarrays
DNA microarrays can be used to compare
gene expression patterns, multiple genes
at a time:
◆ Which genes are expressed in which
cells and under what conditions.
◆ Which genes are expressed differently
in diseased cells compared to normal cells.
◆ Which genes are expressed differently
when a patient is administered a drug. 10
Raw array
speckles
(B)
(A)
Light
Background
(C)
Shape
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Technology differences
◆ Pin spotting or inkjet printing or
photolithography
◆ 1-sample or multi-sample
◆ Almost-complete sequences (cDNA)
or subsequences (oligoneucleotides)
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Processing steps
Spotted
Image
4000
2000
Probability Interv
6000
8000
Statistical
Analysis
0
Raw
Image
0
100
200
300
Genes
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