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 microarray Control spots Evaluation of an array setup By H. Bjørn Nielsen Array 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-hours Spotted arrays  2-6 Months Affymetrix  1-2 weeks Costs after setup Cost pr. experiment Affymetrix  USD 500 (chip) (sample prep.) Spotted arrays  USD (slide, 4000 spots) + 70 (sample prep.) Cost pr. spot / transcript Spotted arrays  USD 0.10 / 0.10 Affymetrix  USD / Costs

Choice of Technology Flexibility To 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 Quality Reproducibility of data (Pearson correlation) Affymetrix  0.95 Spotted arrays  (highly variable) Sensitivity Affymetrix >1:100,000 (linearity) (Affymetrix.com) Spotted (oligos)  1: (linearity) (Ramakrishnan et al. 2002) Specificity Most studies show: decreased specificity with oligonucleotide length i.e. 50mers gives false signal down to 75% permutation 30mers down to 90%. (Kane et. al.2000; Ramakrishnan et al. 2002) Data quality

Choice of Technology Required material Affymetrix  A pre-designed chip from Affymetrix Spotted oligonucleotide  Sequence information Spotted cDNA  cDNA library Required materials

comparisons AdvantagesDisadvantages PCR products Inexpensive to setupContamination problems Hard to design probes Unequal amplification of probes Plasmids (RCA) Inexpensive to setup Uniform amplification No design opportunities Contamination problems Plasmid may disturb signal Oligos Designable probes Normalized probe concentrations Inexpensive pr. experiment High setup costs App. USD. 20 per oligo Affymetrix High reproducibility Fast to set up Inexpensive pr. probe High setup cost App.250,000 USD Arrays 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 clones Produce cDNA library, sequence the clones and re-array PCR Product purification and control (gel) Dilute in buffer (DMSO/SSC) Spot on solid surface Bind to surface setup cDNA array

Aspergillus oryzae array example

All controls are printed in each quadrant of the array i.e. by each of four print pins Incl. controls for Local and global reproducibility Control for slide saturation “Housekeeping” genes Spike-in controls Negative controls Pin washing controls Non-specific binding PCR primer binding Vector binding (pYes) Poly-A control Control spots A. oryzae example

Gamma-Actin:100%50%25%12.5%6%3% Glyceraldehyde-3-Phosphate Dehydrogenase H 2 O 50% DMSO Salmon sperm DNA PCR mix + primer pYes vector Empty Poly-A 100% DMSO Control field A A. oryzae example

In realitySaturated spots (ideal) ,563 Spot saturation A. oryzae example

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 Protein Tubulin Beta Glyceraldehyde 3- P. Dehydrogenase 40S Ribosomal Prot. Galactanase H.i Arabidopsis CAB Arabidopsis RCA Arabidopsis rbcL Human COT-1 Fibrin Tubulin Alpha Actin-Rela. Prot. Alpha-Amylase Tubulin Alpha Ca-Indep. Phos. Lipase 40S Ribosomal TFIID Golgi Memb. Prot.-Sort Prot. Spindel Assembl. Checkpoint Prot. Control field B A. oryzae example

Indirectly Absolute Directly or Indirectly comparable data

Direct ratio:  gal i + /  wt i Indirect ratio: Mean (gal i + / wt i ) Directly or indirectly comparable data

Log 2 (intensity) Log 2 (Ratio) Reproducibility - same sample in both channels

Glyceraldehyde-3-Phosphate Dehydrogenase Theoretical Gaussian Quantiles Sample Quantiles Is Data Gausian Q-Q plot