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