data analysis of dna microarrays - amazon s3...data analysis of dna microarrays can we detect...
TRANSCRIPT
![Page 1: Data Analysis of DNA Microarrays - Amazon S3...Data Analysis of DNA Microarrays Can we detect knockdown of gene expression using DNA microarrays? sample1 sample2 Starting with two](https://reader036.vdocument.in/reader036/viewer/2022081405/5f08a6547e708231d4230d3b/html5/thumbnails/1.jpg)
Data Analysis of DNA Microarrays
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Can we detect knockdown of gene expression using DNA microarrays?
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sample2sample1
Starting with two biological samples
WT KO
RNA RNA
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WT KO
Green=cy3Red=cy5
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Spotted microarrays
www.molgen.mpg.de
Signal is average of pixel intensities of spot
2 numbers per spot
Red=500Green=100Red/Green=5 (5 Fold Greater)
Microarray Measurements
cy5
cy3
Signal: Spotted arrays
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Agilent scanner image Feature
extractor Txt filemicroarray
Processing microarrays: Scanning and Image analysis
File is largeNeed to truncate
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Spot Intensity: Mean or Median?
Which is more affected by extremes?Which is better estimate of spot intensity?
Mean=60,000Median=60,000
Mean=70,000Median=60,000
Image
All pixels of a spot are used to calculate a Mean or Median Intensity
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Subtracting Background
Background=50
Spot Signal=60,000
“True” Signal=60,000-50
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Reduce number of rowsRemove rows 1-21
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Copy columns A, G, I, J, K, L**, AI, AR, AS into new sheet
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Create background subtracted column for red and green signals
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Clean file of excess probe information
Select AllSort by gene name
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Delete unused negative and positive control: eQCs
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Delete unused negative and positive control: eQCs
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Delete unused negative and positive control: eQCs
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Within-Slide Normalization
Normalization balances red and green intensities. Imbalances can be caused by
Different incorporation of dyesDifferent degradation of dye
In practice, we usually need to increase the red intensity a bit to balance the green
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562 nm
cy3
cy5
www.amersham.com
cy3 cy5
Light sensitivity: cy5 more easily degraded
664 nm
510 nm
emission
cy3 and cy5: Commonly used dyes
cy3 cy5
~700
~500
emission
cy3
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Let’s begin the normalizationprocess:
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Create mean signal to estimate dye bias
Green backgroundcorrected
red backgroundcorrected
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Create meCreate mean signal to estimate dye bias
1.7 FC difference
green red
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Normalize your lower abundant channel(increase) by factor to have meanExpression across the array equal
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Spotted microarrays
www.molgen.mpg.de
2 numbers per spot
Red=500Green=100Red/Green=5 (5 Fold Greater in Red)
Red=100Green=500Red/Green=0.2 (5 Fold Less in Red)
Calculating Differences in Gene Expression
Log2(5)=~+2Log2(0.2)=~-2
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Compare expression of each Channel (using normalized channelIn one condition)
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Calculate log2 ratio of each chann
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What happened??
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Correct for negative intensity
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And NOW to the fun…
How many genes were differentially expressed between your 2 samples?
Was the expression of your gene of interest significantly changed between the two samples?...can we assess this directly
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Create scatter plot of log2 ratios (green versus red)
Create Scatter Plot
log2 red vs green
-10
-8
-6
-4
-2
0
2
4
6
8
10
0 2000 4000 6000 8000 10000 12000log2 red vs green
2 fold increase
2 fold decrease
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Distribution of log2 ratios
What are we expecting????What color would all of these spots be??
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How many genes on the array?
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Trends in Data
How many changes do you see?What could these changes mean?How can we find out more about these genes and their functions? Which biological processes are up-regulated, down-regulated, no change?
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Good luck!!