How To Develop A Microarray

Transcription

1 Integrated microarray solutions at the CPGR in Cape Town, South Africa Reinhard Hiller, PhD Director Tecan Workshop Sep 2007

2 CPGR

3 CPGR in a nutshell CPGR members Infrastructure Expertise Services Collaborative projects Non-exec directors ExCo CPGR board Funding CBT, PB; MRC Mission Collaborative projects Service & R&D organisation SOP-based services stimulate excellent research (IP), Knowledge base in Genomics & Proteomics Boost biotech economy

4 CPGR platform perspective Core focus area (e.g. Plant Genomics) Capacity development Collaborations New Commercial ventures

5 biocompatible biocompatible ht ht protein protein purification, purification, MALDI- MALDI- TOF/TOF-based TOF/TOF-based protein protein id id & de-novo de-novo sequencing, sequencing, abundance abundance & functional functional protein protein microarrays microarrays Target Target validation validation on on ht ht qrt-pcr qrt-pcr platform platform & TaqMan TaqMan low-density low-density arrays arrays CPGR: An integrated core technology platform CPGR: An integrated core technology platform Affymetrix Affymetrix GeneChip GeneChip system system for for large-scale large-scale genotyping genotyping & & RNA RNA expression expression profiling profiling High-quality High-quality custom custom DNA DNA microarraying microarraying & analysis analysis

6 Project / service pipeline: July 2007 Concept Preparation In progress Finished n=19 n=39 n=16 n=4 Discontinued Biomedical Plant Biotech Bioprospect. Veterinary 20% 23% 5% 4% 48% Preparation Concept In progress Finished Disco ntinued

7 Project / service pipeline: July 2007 Clinical trial 0% Pharmaceutical 0% Tb 15% Grape 2% CVD 2% HIV 2% Maize 2% Mouse 2% NAFLD 2% Parasites 2% Yeast 6% Clinical trial Pharmaceutical Grape CVD HIV Maize Mouse Asthma 4% NAFLD Parasites Cancer 15% Yeast Asthma Various 11% Diagnostics 13% Diagnostics Drug discovery Biomarker Malaria 9% Biomarker 9% Drug discovery 4% Malaria Various Cancer Tb

8 Workflows

9 Analytical work-flows in GENOMICS Discovery Validation RNA expression & DNA genotyping microarrays Custom microarrays & ABI 7900 TaqMan system Affymetrix GeneChip platform Other customized commercial arrays, e.g., Agilent Custom oligo- & cdna manufactur -ing Custom DNA microarrays TaqMan RT- PCR & genotyping & mirna assays High-density arrays Sequenced organisms Non-model organisms cdna arrays Custom development Focussed validation of largescale genomic / transcriptomic data Low-density arrays and / or RT-PCR assays High genome coverage High quality standards (e.g., Agilent bioanalyser) High reliability (e.g., Tecan hyb station) High throughput Large sample numbers High quality Low sample requirement Low costs

10 DNA microarray applications at CPGR DNA RNA CGH SNPs ChIP on chip Gene expression Splice variants µrnas Chromosome Gene Promoters mrna Exons µrnas Copy-number analysis Genotyping Regulatory assessment Gene regulation Differential splicing RNA regulation Affy Agilent NimbleGen Operon Affy Agilent ABI 7900 Affy Agilent Affy Agilent Operon ABI 7900 Affy Exiqon BioPlex ABI 7900

11 (DNA) microarray workflows at CPGR Liquid-handling robot Spotter Hyb station Scanner Through-put Cost-efficiency Quality Compatibility with other (microarray) applications

12 Integrated Proteomics at CPGR Sample Extraction/ purification Processing Electrophoresis Chromatography Separation Fractionation Identification Concentration Quantification 1D/2D HPLC Protein profiling/imaging Mass Spectrometry (IMS) Digestion Chromatography Protein microarrays Protein expression Shotgun MS/MS itraq icat m/z MS/MS Peptide IDs Sequence Peaks / proteins of interest MALDI-TOF/TOF Suspension arrays

13 Protein microarray development Allergen A Allergen B Allergen C Allergen D Eczema Rhinitis Asthma Extract Component(s) Microarray (Diagnostic) profile

14 Side-step: Decreased costs via increased throughput 4 assays per slide 36 slides per run 36 Slides á 4 assays á 80 allergens = 144 IgE assays per run = > reactions per run IgE & IgG (IgG4) = > reactions per run 4 Slides á 16 assays á 80 allergens = 64 assays per run (= 4 slides) = > reactions per batch IgE & IgG (IgG4) = > reactions per batch

15 Examples

16 Allergen Allergen molecule molecule Allergen-triplet Allergen-triplet after after fluorescence-reaction fluorescence-reaction Fluorescence-labelled Fluorescence-labelled secondary secondary anti-human anti-human IgE IgE antibody antibody IgE-antibdy IgE-antibdy (in (in patient spatient sserum) serum) Biochip: Biochip: Modified Modified glass glass slide slide (25x75 (25x75 mm) mm) (To (To date) date) allergens allergens microarrayed microarrayed in in each each reaction reaction site site (4 (4 identical identical copies) copies) 44 reaction reaction sites sites (3 (3 samples samples control) control)

17 Manual allergen microarray assay Sample: Venous / capillary serum Prepare Allergen Microarray Incubate Sample Wash Allergen Microarray Incubate Secondary Antibody Duration: 3 h Wash Allergen Microarray Scan Microarray(s) Analyse Microarray Image(s) Duration: 10

18 Multiplex diagnostics: Component-resolved diagnosis

19 IgE profiles in asthmatic children (2006) Hiller, Jeebhay, Lopata & Naidoo, manuscript in preparation

20 (Allergen) microarray assay automation Diagnostics Epidemiological studies R&D (profiling) (Diagnostics) R&D Validation

21 (Seafood) allergen characterisation Allergen profiling Allergen discovery Diagnostics Allergen detetection Ab assays for allergen detection (bead assays) Reactive bands Immunoblots MTP Affinity capturing Beads Microarrays HPLC Columns Cell culture Diagnostic assays for workers in the seafood industry - > better prevention & treatment of occupational allergies MS New (and expected) allergens in seafood species

22 Array-based allergen profiling Extraction Allergenic molecule Non-Allergenic molecule 6 mm Fractionation 6 mm

23 Identification / characterization of latex allergens Screening with 200 samples from latex allergic patients Pearson correlation (> 0.85, p > 0.05) Pre-characterize unknown fractions Information gained: Pre-characterized allergens based on cross-reactivity towards homologous proteins Information about composition of fractions plus state of allergens = important for manufacturers of immunotherapy reagents (extracts)

24 Allergen microarrays & immunotherapy development IgE concentration (FI values) Allergen concentration in solution Which biological source contains the immunodominant allergen suitable for immunotherapy?

25 Screening for immunodominant allergens & epitope profiling ng / ml nole e 2 rbet v 2 rcyn d 12 rhel a 2 rhev b 8 rmer a 1 rpar j 3 rphl p 12 rpho d 2 Bet v 2 (birch profilin) contains all the epitopes of 8 homologous profilins Therapy candidate µg 5 pg / ml Harwanegg & Hiller, unpublished results

26 Microarray-based B-cell epitope profiling: Tb peptide arrays Whole proteome Peptide synthesis Protein a, b, c c Linear ab binding epitopes Diagnostic peptides / signatures Therapeutic targets Peptide microarray

27 Manual vs automated Tb peptide arrays Manual Automated Manual Automated

28 Significant increase in S/N in automated assays Increase in foreground signal using HSPro slide batches Increase in the proportion of spots detected above cut-off levels 100% % values expressed as percentages batch 793 manual batch 793 HS4800Pro batch 866 manual batch 866 HS4800Pro 80% 70% 60% 50% 40% 30% 20% 10% batch 866 HS4800Pro batch 866 manual batch 793 HS4800Pro batch 793 manual 0.0 F635 Median - B635 F635 Total Intensity 0% % Spots with SNR>3 Signal - background Total signal % spots above cut-off Secondary Antibody Incubation Precision (within & between slide & between runs, batch-to-batch etc) Specificity (comp. inhibition assays)

29 Other microarray applications Operon Agilent Exiqon JPT Dual malaria parasite DNA microarrays (HS 4800) 1-plex and 4-plex genotyping arrays (CGH!) (HS 4800) MirCury mirna arrays -> mirna biomarker focus area in South Africa (HS 4800) Tb peptide arrays; CMV arrays; infectious disease arrays (malaria); allergen epitope profiling (HS 4800) VBC-GENOMICS Gentel CPGR Allergen microarrays (HS 4800) Cytokine antibody arrays (Hydroflex); cancer biomarker arrays (HS 4800) Arrays of fractionated Tb cultures; tissue extract arrays; functional protein arrays (p450, p53) (HS 4800 & HydroFlex)

30 Translational issues Biomarker/MDx development GAP Biology Pattern Signature Better diagnostics Novel drug targets Novel therapeutic applications More efficient treatments Personalized medicine Early discovery / pre-clinical development: min. 2 years Validation

31 Acknowledgements CPGR Jonathan Blackburn Anne Brill Rachel van Dyk Jo McBride Maré Vlok Kutala Pangwa Scientists Mark Bowmaker (SA) Mohamed Jeebhay (SA) Andreas Lopata (SA) Rajen Naidoo (SA) Companies JPT (Germany) VBC-GENOMICS (Austria) Christian Harwanegg Funding Cape Biotech Trust (CBT) PlantBio (PB) Medical Research Council (MRC)