EXPERIMENTAL DESIGN Experimental and Pre (Quasi) Experimental Designs.
Experimental Design and Setup. Experimental Design What is the question? Which experiments will give...
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Transcript of Experimental Design and Setup. Experimental Design What is the question? Which experiments will give...
Experimental Design and Setup
Experimental Design
What is the question?
Which experiments will give the answer?
How many replicates do we need?
•Choice of technology
•Setting up a microarrayControl spotsEvaluation of an array setup
By H. Bjørn NielsenArray Setup and Evaluation
Affymetrix
Spotted
Choice of Technology
-Costs
-Flexibility
-Data Quality
-Required material
Affymetrix or spotted arrays
Affymetrix
Spotter
Choice of Technology
Facility setup cost Equipment
Affymetrix USD 250,000 Spotted arrays USD 70,000 + ( USD 20 pr. oligo)
Man-hoursSpotted arrays 2-6 MonthsAffymetrix 1-2 weeks
Costs after setupCost pr. experiment
Affymetrix USD 500 (chip) + 200 (sample prep.) Spotted arrays USD 30-50 (slide, 4000 spots) + 70 (sample prep.)
Cost pr. spot / transcriptSpotted arrays USD 0.10 / 0.10Affymetrix USD 0.003 / 0.04-0.07
Costs
Choice of Technology
FlexibilityTo design custom arrays
- Spotted arrays > Affymetrix
To re-design arrays
-Affymetrix offers a series of pre-designed arrays, but custom designs are expensive
-Spotted arrays are expensive to re-design
Flexibility
Choice of Technology
-Data QualityReproducibility of data (Pearson correlation)
Affymetrix 0.95 Spotted arrays 0.80-0.95 (highly variable)
SensitivityAffymetrix >1:100,000 (linearity) (Affymetrix.com)Spotted (oligos) 1:300.000 (linearity) (Ramakrishnan et al. 2002)
SpecificityMost studies show: decreased specificity with oligonucleotide lengthi.e. 50mers gives false signal down to 75% permutation30mers down to 90%. (Kane et. al.2000; Ramakrishnan et al. 2002)
Data quality
Choice of Technology
Required materialAffymetrix A pre-designed chip from AffymetrixSpotted oligonucleotide Sequence informationSpotted cDNA cDNA library
Required materials
comparisons
Advantages Disadvantages
PCR products
Inexpensive to setup Contamination problemsHard to design probes Unequal amplification of probes
Plasmids (RCA)
Inexpensive to setupUniform amplification
No design opportunitiesContamination problemsPlasmid may disturb signal
Oligos Designable probesNormalized probe concentrationsInexpensive pr. experiment
High setup costs App. USD. 20 per oligo
Affymetrix High reproducibilityFast to set upInexpensive pr. probe
High setup cost App.250,000 USDArrays only available for limited number of species
Array technology
- What collection of genes do we want to measure
What kind of information are we looking for
How are we going to normalize the data afterwards
Which controls do we need/want
considerations Array layout
Oligonucleotide array
Oligonucleotides synthesis
Purified (HPLC)
Normalize concentration
Dilute in buffer (DMSO/SSC)
Spot onto solid surface by array robot
Bind to surface
setup
Typically don by
external company
Collect relevant clonesProduce cDNA library, sequence the clonesand re-array
PCR
Product purification and control (gel)
Dilute in buffer (DMSO/SSC)
Spot on solid surface
Bind to surface
setupcDNA array
Aspergillus oryzae arrayexample
All controls are printed in each quadrant of the arrayi.e. by each of four print pins
Incl. controls forLocal and global reproducibilityControl for slide saturation“Housekeeping” genesSpike-in controls
Negative controlsPin washing controlsNon-specific bindingPCR primer bindingVector binding (pYes)Poly-A control
Control spotsA. oryzae example
Gamma-Actin: 100% 50% 25% 12.5% 6% 3%
Glyceraldehyde-3-PhosphateDehydrogenase
H2O
50%
DM
SO
Salm
on s
perm
DN
APC
R m
ix +
prim
erpY
es v
ecto
rEm
pty
Poly
-A
100%
DM
SO
Control field AA. oryzae example
100 50 25 12,5 6 3
In realitySaturated spots (ideal)
100 50 25 12,5 6 3
Spot saturationA. oryzae example
0,00
0,05
0,10
0,15
0 0,05 0,1 0,15 0,2
Global std
Local std
Local vs. Global Variation Glyceraldehyde-3-Phosphate Dehydrogenase
DNA-Dependent RNA Polymerase II
Gamma-Actin
Histone H2A
Beta-Actin
ER Chaperone BIP
60S Ribosomal ProteinT
ubulin Beta
Glycerald
ehyde 3-P
. Deh
ydrogenase
40S Ribosomal Prot.
Galactanase H.i
Arabidopsis CAB
Arabidopsis RCA
Arabidopsis rbcL
Human COT-1
Fibrin
Tubulin Alpha
Actin-R
ela.P
rot.
Alpha-Amylase
Tubulin Alpha
Ca-Indep. Phos. Lipase
40S Ribosomal
TFIIDGolgi Memb. Prot.-Sort Prot.
Spindel Assembl. Checkpoint Prot.
Control field BA. oryzae example
1 3
2 4
Indirectly Absolute
1 3
2 4
Directly or Indirectlycomparable data
-3
-2
-1
0
1
2
3
-3 -2 -1 0 1 2 3 4
Direct ratio: gali+/ wti
Indirect ratio: Mean (gali+/ wti)
Directly or indirectly comparable data
-10
-8
-6
-4
-2
0
2
4
6
8
10
8 9 10 11 12 13 14 15 16
Log2(intensity)
Log 2
(Rat
io)
Reproducibility- same sample in both channels
Glyceraldehyde-3-Phosphate Dehydrogenase
Theoretical Gaussian Quantiles
Sa
mp
le Q
ua
ntile
s
Is Data GausianQ-Q plot