Experimental Design May 2007 Hanne Jarmer. What is Experimental Design? Question Answer Data...
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Experimental DesignMay 2007
Hanne Jarmer
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What is Experimental Design?
Question
Answer
Data Analysis
Experiments
Often not the final answer!
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What do we need?
• What is the question(s)?
• Which experiment(s) will give the answer?
• How many replicates do we need?
The “correct” experimental design?!
• Several equally good designs
• Balancing
• Replicating
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The Question
What is a good question?
... that can be answered by the use microarray experiments?
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The Questions
Why do obese children have diabetes more frequently?
What is the connection to fitness/diet?
?
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The Experiments
1. From question: Define your categories
2. Access to material? (living human brain?)
3. How many replications?
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The Experiments
Determine from where you’d get variation?
Hopefully: Biology !!
- array
- spot
- time
- patient gender/age/other
- dye
- technician
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The Experiments
From question: Define your categories
Sick < > healthy
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The Experiments
Access to material? ... Mmmm, three of these ...
Human samples not always possible ...
Maybe rats instead?
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The Experiments
How many replications?
Impossible to answer!
Depends on how ...
... strong the biological signal is
... much you’d like to be able to trust the result
... you plan to analyze
... much you can afford ...
Biological replications are always preferable!
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The Experiments
Variation ...
How do we get (only) what we want?
THE KEY: BALANCING .. all other sources of variation
SICK MEN HEALTY MEN
2 old, 6 young 2 old, 6 young
3 day A, 3 day B 3 day A, 3 day B
2 batch 1, 4 batch 2 2 batch 1, 4 batch 2
Technician Y Technician Y
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DYE SWAP ... is it really necessary?
The non-linear normalization should take care of this ... right?!
This is mostly true,... but not always:
“Technical” or “biological” dye swap?
A biological replication always give you more !!
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The Experiments
Important: Pick conditions that:
... will maximize the interesting biological signal
Ex.: Wild-type bacteria < > TF-mutant
... and ...
choose a growth medium where the TF is active!!
TF
List of genes?
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The Experiments
Important: Pick conditions that:
... will maximize the interesting biological signal
... while minimizing the overall difference
Less optimal: Liver < > Brain
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Basic Designs
“Reference design” “Loop design”
Stanford-type microarrays:
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Basic Designs
“Loop design”
2 samples from the same category
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Please, take home...
1. Question How to save the world?
2. Categories A - B -C
3. Balance
4. Replication (biological)
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Example: Why not AIDS?
1. Question: Why do some HIV-infected ... not develop AIDS?
2. Categories:H
IV-i
nfec
ted
Patient type
0100
10 11
0 = no AIDS
1 = AIDS
0 = no HIV
1 = HIV
NEVER AIDS
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Example: Why not AIDS?
Time
Blood samples
Blood samples
HIV
AIDS
00
01
10
11
~ 20 years
HIV
-inf
ecte
d
Patient type
0100
10 11
0 = no AIDS
1 = AIDS
0 = no HIV
1 = HIV
Determination of categories
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Example: Why not AIDS?
Balancing and replicating
Variation can come from:
Village, age, doctor, .... TH-cell extraction
Relatively few “0” patients ...
- Pick corresponding “1” patients
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Example: Why not AIDS?
HIV
-inf
ecte
d
Patient type0 = no AIDS
1 = AIDS
0 = no HIV
1 = HIV
01
11
00
10
3 P-values for each gene ...
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Example: Why not AIDS?
The 2-way ANOVA results:
3 lists:
- Patient-type difference
- +/- HIV difference
- The Interaction:
“0”-patient genes that behave
unexpectedly during HIV-infection
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Example: Why not AIDS?
- CCR5-mutation (no HIV)
- The golden combination of HLAs (HLA-B57 in particular)
- Something unknown !?
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Please, take home...
1. Question How to save the world?
2. Categories A - B -C
3. Balance
4. Replication (biological)
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Short Discussion
A) Questions:
1) How will Treatment A or B or the combination of both affect the genes in the malaria parasite?
2) Will either affect the production of the “sticky” proteins responsible for the aggregation of the red blood cells?
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Short Discussion
B) How would you design this study?
C) Which categories?No treatment
Treatment A
Treatment B
The combination
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Short Discussion - in groups
The study is done using two-color microarrays and 4 biological replications
D) Which samples would you label red and which would you label green?
E) What will you hybridize with what?
N-1 A-1 B-1 AB-1
N-2 A-2 B-2 AB-2
N-3 A-3 B-3 AB-3
N-4 A-4 B-4 AB-4
N-1 A-1 B-1 AB-1
N-2 A-2 B-2 AB-2
N-3 A-3 B-3 AB-3
N-4 A-4 B-4 AB-4
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Short Discussion - in groups
N-1
B-1
A-2
AB-2
N-3
B-3
A-4
AB-4
N-2
AB-3
A-3
B-4
AB-1
N-4
B-2
A-1
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Short Discussion - in groups
A) Can you come up with another interesting biological question?
B) Which categories would you need?
C) How would you balance this study?