© fimm - institiute for molecular medicine finland high throughput biomedicine unit (htb) at fimm...
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© FIMM - Institiute for Molecular Medicine Finland www.fimm.fi
High Throughput BiomedicineUnit (HTB) at FIMM Technology Centre
www.fimm.fi
FIMM Technology Centre
High-throughput Biomedicine
Biomarker validation
Metabolomics
Genomics & Bioinformatics
IT
Imaging & clinicalinformatics
State-of-the-art equipment
~ 50 Technology experts-PIs-Senior scientists-Post Docs-Masters-Bioinformaticians-Lab technicians
National and international research core unit providing an extensive spectrum of biomedical research services.
High-throughput Biomedicine
www.fimm.fi
High density format
96 well plateVol/well= 100ul 1536 well plate
Vol/well= 4 ul
Microarray format
384 well plateVol/well= 25ul
What is needed for a HTS?What is needed for a HTS?
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High density format
High throughput reader
What is needed for a HTS?What is needed for a HTS?
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What is needed for a HTS?What is needed for a HTS?
High density format
High throughput reader
Data analysis
www.fimm.fi
HTB Equipment
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BeckmanCoulter integrated robotic system-used for splitting libraries on Labcyte ECHO source plates, by pipetting-running fully automated screens with cell or biochemistry based assays
Motoman robotic armBiomek FXp pipetting robotMultidrop Combi (x2) and Combi nl (x1)Cytomat 6001-plate incubator for cellsPlatelock-plate sealerV-spin-centrifugeDelidding stations and plate hotels on robot deckParadigm plate readerCytomat 24 plate hotel
Labcyte Access robotic system-used for creating assay plates with chemicals and/or siRNAs-miniaturized cell-based and biochemical screening
Labcyte robotic armLabcyte 550 Omics2 Screening2 (2.5 nl droplet)Echo 525 (25 nl droplet)Labcyte LX bulk filler (4 source liquids)Xpeel-plate peelerPlatelock-plate sealerV-spin-centrifugeDelidding stations and plate hotels on robot deck
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Collaborations
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550 525 550
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RNAi screening Chemical screening Microarray printing
High Throughput Biomedicine UnitHigh Throughput Biomedicine Unit
Sharing of chemicals with
academic groups
•siRNA screens
•miRNA screens
•Combination screens•of siRNA, miRNA, chemicals
•Drug Sensistivity and Resistance Testing
(DSRT)
•Chemical Screens
•Biochemical Assays
•Reverse Phase Protein Lysate Array (RPPA)
•Serum printing
•Oligonucleotide printing
Personalized medicine
ex vivo testing of patient Cells with oncology drugs:
LeukemiaOvarian cancerGlioblastoma
Assay miniaturisation
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1. RNAi screening1. RNAi screening
Screening is mainly performed on 384-well plates (96-well possible)Any adherent cell-line is applicableScreening volume/ sample 25 ul1 plate: 320 samples + 64 controls
Readout: high content microscopy, live cell microscopy or cell viability
Analysis: image analysis, statistical analysis, pathway analysis
siRNA library: Ambion Silencer Select full genomeCustom made library: Qiagen or other vendors
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Ambion SilencerAmbion Silencer® ® SelectSelect Human siRNA Human siRNA LibraryLibrary V4V4
Nuclear hormones (47)
Ion channels (338)
Proteases (494)GPCR (380)
Kinases (710)Phosphatases
(298)
Druggable genome (9032)
Extended Druggable genome (10 415)
Human Genome Collection (21 585)
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Incubation 72 h-7 days at 37oCDispensing of detection reagent on cells
Luminescence / intensity readout
Cell culture 384 well assay plates
1) Dispensing of transfection reagent on assay plates 2) Dispensing of cells on the plates
siRNA Screen WorkflowsiRNA Screen Workflow
siRNA libraries on 384 well ECHO plates
siRNA administration (nl)
A
B
Fixing and staining of cellsMicroscopy readout
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High Content ScreeningHigh Content Screening
Image analysisNumbers
cells
Picture
Microscope
Microtiter plate
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2. Chemical Screening2. Chemical Screening
• Drug sensitivity and resistance testing (DSRT)
• Custom screens (mammalian, yeast, prokaryotes)
• Biochemical screens
• Chemical library combined with siRNA KO
Screening is mainly performed on 384-well plates (1536 well possible)Cell-lines, patient cells, suspension cells...Readout: can be chosen among several plate readers, microscopy
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HTB chemical LibrariesHTB chemical Libraries
› HTB Unit has access to several chemical & genomic libraries:
306 FIMM and NIH National Cancer Institute oncology drug collection
446 NIH Clinical collection 2000+640 Microsource and ENZO, including FDA approved drug
collection and natural products 1120 Tocris Tocriscreen mini 2500 NIH NCI (National Cancer Institute) collections 6000 Tripos Structures collection 15 000 ChemDiv Peptidomimetics 25 000 ChemDiv TP02 and TP03 collections 30 000 ChemBridge DivSet and CNS Set 30 000 Specs Consortium collection
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Cell culture 384 well assay plates
Dispensing of cells on the plates-patient cells-cell lines
Drug administration (nl)
Incubation 72 h at 37oCDispensing of detection reagent on cells-cell viability mesurement
Luminescence readout
Drug sensitivity and resistance testing (DSRT)Drug sensitivity and resistance testing (DSRT)
Oncology collection 306 drugs5 conc. 10,000-fold conc. range
300 dose response curves
Effective drugs
Resistant drugs
Microscopy readout under development
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What is in the FIMM oncology collection? What is in the FIMM oncology collection?
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Currently 306 active substances:
• Conventional chemotherapeutics
• Immunosuppressants
• Tyrosine kinase-type inhibitors• Abl, Src, EGFR, FGFR, VEGFR, JAK,• IGF1R, PDGFR, Met, ALK Kit, Flt3….
• S/T-type inhibitorsAurora, PLK1, MEK, TTK, PDK1, Akt, Wee1, PKCs, Cdks, Chk1…
Scattered layout for controls:•Benzethonium chloride for low control•DMSO for high control
Algorithms and scripts are being developed to QC and analyse the data
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3. Microarray Printing3. Microarray Printing
Serum microarray
RPPA
25 mm
75
mm
Oligonucleotide microarray
We hope to use the ECHO in the future to print arrays (Echo Array Maker software)
Reverse Phase Protein Array (RPPA)Serum samplesOligonucleotide arrays
Aushon 2470 contact printer
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Cells on plates, Incubation 72h Cell lysis
Heating of the plates at +95C
Spotting with Aushon 2470
SyproRuby staining of the slides
Scanning the slides for tot. protein
Staining the slides with antibodies
Scanning the slides with Li-Cor Odyssey
Analysing the scans using Array-Pro Analyzer
Transfer of the lysates on the arrayer compatible plates
Reverse Phase Protein Array Reverse Phase Protein Array (under development)(under development)
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•Preplating of chemicals on assay plates with ECHO
•Distributing of single aliquots to different research groups
•Partner in the DDCB network, a national infrastructurefor drug discovery and chemical biology research in
Finland
Sharing of chemicals with academic groupsSharing of chemicals with academic groups
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1. You have an idea for a screen
2. First planning meeting-To estimate the feasibility of the project, to give hints about assay development.
3. Assay development
4. Assay robustness and quality testing-The assay needs to fill quality measurements (e.g. Z’-value, signal-to-noise ratio)
5. Second planning meeting-To estimate wether screening can be started, planning the scedule
6. Screening-pilot screen-primary screen-(counter screen)
7. Data analysis-identification of hits, pathway analysis, statistical analysis
8. Validation-required for publications
OverviewOverview of the screening process of the screening process
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Some statistics of last year:Some statistics of last year:
•392 DSRT sets (627,200 samples)
•150 Custom designed chemical plates (48,000 samples)
•A large chemical screen with 100,000 compounds for an international pharma company
•2 full genome siRNA screens with image analysis (64,755 siRNAs each)
•~300 plates of custom designed siRNA/miRNA + compound screens (96,000 samples)
•546 distributed compound aliquotes
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Published papersPublished papers
Antila H. et al. 2014. Utilisation of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells. J Neurosci Methods
Stylinaou M. et al. 2013. Antifungal application of non-antifungal drugs. Antimicrob Agents Chemother.
PemovskaT. et al. 2013.Individualized Systems Medicine (ISM) strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery
Tang J. et al. 2013. Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways. PLOS Computational Biology
Nybond S. et al. 2013. Antimicrobial assay optimization and validation for HTS in 384-well format using a bioluminescent E. coli K-12 strain. EUFEPS
Denisova O.V. et al. 2012. Obatolax, Saliphenylhalamide, and Gemcitabine Inhibit Influenza A Virus Infection. J.Biol. Chem.
Koskela H.L.M. et al. 2012. Somatic STAT3 Mutations in Large Granular Lymphocytic Leukemia. NEJM
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FIMM High Throughput Biomedicine Unit FIMM High Throughput Biomedicine Unit
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Evgeny Kulesskiy (Graduate stud.)
Karoliina Laamanen (MSc)
Anna Lehto (BSc)
Vilja Pietiäinen (PhD)
Swapnil Potdar (MSc)
Jani Saarela (PhD)
Contact Details:Contact Details:
Chemical screening, DSRT: [email protected]
siRNA screening, imaging: [email protected]
Unit Head:[email protected]
Carina von Schantz-Fant (PhD)
Laura Turunen (Lic.Sc.)
Heidi Virtanen (PhD)
Krister Wennerberg (PhD)
Päivi Östling (PhD)