The Bio-Screening Core Facility PETER BANKS PETER.BANKS@NCL.AC.UK CARMEN MARTIN-RUIZ CARMEN.MARTIN-RUIZ@NCL.AC.UK
Bio-Screening Facility Information High Throughput Screening Facility Biomarker Facility Peter Banks Carmen Martin-Ruiz Peter.Banks@ncl.ac.uk Carmen.Martin-Ruiz@ncl.ac.uk Claire Kolenda Claire.Kolenda@ncl.ac.uk Craig Parker Craig.Parker@ncl.ac.uk Cookson/Leech Building Ageing Research Labs Medical School Campus for Ageing and Vitality https://www.ncl.ac.uk/htsf/ https://www.ncl.ac.uk/ion/about/facilities/biomarkers/
High Throughput Screening Combine robotics, high throughput reagents and expertise to provide a bespoke cell based screening service to all researchers in FMS ◦ Laboratory Automation ◦ Genetic screening services for a range of organisms ◦ Drug screening in collaboration with LifeArc
Equipment 2 sites – 2 nd floor Cookson and 2 nd floor Leech Liquid handling mammalian cells ◦ Beckman FX within a sterile enclosure – Class II ◦ Beckman FX – Class I Colony Pinning microbes ◦ S&P BM3 ◦ S&P BM5 ◦ Singer ROTOR Data collection ◦ Agar plate imaging systems ◦ Automated plate readers ◦ High Content Imaging system – Bioimaging ◦ High Throughput Flow Cytometer – FACS Ancillary ◦ Agar plate pouring ◦ Multi well plate filling
Genetic Screening Cell based genetic screening is the combination of Yeast collections laboratory automation with high throughput ◦ Deletion library ~4500 strains biological reagents. ◦ Overexpression library ~6000 strains ◦ Identify new research directions ◦ DaMP Library ~800 strains ◦ Preliminary data generation for grant applications ◦ Histone point mutant library ~ 400 strains ◦ Temperature sensitive collections Bacterial collections High throughput reagents are specifically designed ◦ Bacillus subtilis for automation ◦ Escherichia coli ◦ Excellent coverage for large scale experiments ◦ Post screen validation is critical to success Dharmacon Human siRNA library
Dharmacon siRNA Library ◦ G Protein Coupled Receptors Dharmacon siGENOME SMARTpool siRNA library ◦ Protein Kinases -3 plates ◦ siGENOME – type of siRNA ◦ Ion Channels ◦ 4 siRNAs to a single gene in one well – SMARTpool ◦ Phosphatases – 1 plate ◦ ~7500 genes over 28 plates ◦ Proteases ◦ 1nmol in four separate 384 well plates ◦ Ubiquitin Conjugation 1 - Cullins, E1, E2, HECT E3 ◦ Screen 400-2000 plates Ligases ◦ Supply the gene list ◦ Ubiquitin Conjugation 2 - F-box, SOCS box E3 Ligases ◦ Add individual controls to experiments ◦ Ubiquitin Conjugation 3 - RING finger and RING finger-like E3 Ligases ◦ Drug Targets Cherry pick individual 384 or 96 well plates
Drug Screening The HTSF has a collaboration with the life science Index library charity LifeArc to provide FMS researchers with ◦ ~12,000 compounds representative of full LifeArc collection drug screening ◦ LifeArc links basic research to industrial collaborators Kinase library ◦ Expertise – medicinal chemistry, IP and screening ◦ ~6,700 compounds predicted to inhibit kinases ◦ Provided a 20,000 compound library to screen here Natural product library Free to access the libraries ◦ ~1000 purified novel natural products from plants or ◦ LifeArc need to approve the screen fungi Libraries supplied blind FDA approved drugs library ◦ Results must be fed back to LifeArc to deconvolute ◦ ~1,000 compounds 20ul of drug at 10mM in DMSO ◦ 50-200 plates
Case Study – Candida Virulence Developed a high throughput assay for virulence factors ◦ Natural product library ◦ FDA approved drugs library ◦ Preliminary data for a BBSRC grant Screened the 20000 compound LifeArc collection ◦ Initial hit list of ~200 compounds ◦ Many related compounds ◦ Up to 2000 analog compounds
Biomarkers Biomarker: a (molecular) characteristic objectively measured and evaluated as an indicator of normal or pathogenic biological processes An experienced laboratory offering a wide range of procedures to process ◦ Clinical: GCP, GCLP, SOPs, BioCOSHH, HTA ◦ Non-clinical research samples: cell cultures, bird, monkeys, mice, rat... Expertise on the screening of candidate biomarkers Intermediate and flexible capability. “Mid-throughput” Epidemiological value on actual samples
Sample Handling & Technical Support Blood processing and blood cell separation Sampling logistics ◦ PBMC isolation, buffy coats, ◦ Optimal use of blood samples. ◦ PAXgene tubes ◦ Suitability of anticoagulant (or not!) ◦ Cell Preparation Tubes (CPT) ◦ Phlebotomy requirements: blood volumes, timings, priorities ◦ Red cells conditioning Sample tracking Plasma and Serum ◦ Labelling and Barcoding ◦ Pre-treatment and conditioning (anti-oxidants) ◦ Achiever compatible datasets ◦ Aliquoting Coordination with Clinical/Research Teams ◦ RVI/Freeman Labs and CARU ◦ Nationwide and overseas
Follow-up Sample Processing DNA Isolation RNA Isolation ◦ Phenol/Chloroform or Spin columns ◦ PAXgene tubes ◦ Blood samples ◦ Fresh bloods ◦ Tissues ◦ Cell pellets ◦ Cell pellets ◦ Saliva swabs Specialized processing ◦ Blood spot cards ◦ Cryogenic conditioning of cells, cryopreservation ◦ Feathers ◦ Cell culture ◦ Nails ◦ Cytokines: LPS-stimulation, blood cultures DNA & RNA isolation ◦ Flow cytometry ◦ From same sample .... and more ◦ Fresh bloods, tissue ◦ Cell pellets
Biomarker Analysis RNA and Gene expression analysis Novel biomarkers: ◦ RT-qPCR, 384-well capacity, ◦ From cell culture to humans ◦ TaqMan Gene expression assays, ◦ TaqMan MicroRNA assays Novel sources of material: ◦ From humans to animals DNA and Genotyping ◦ Genotyping by Agarose Gels (Mice Ear Notches) Epidemiological scaling: ◦ Genotyping by qPCR (inc.TaqMan™ Genotyping) ◦ From single sample to 384-well plates ◦ Telomeres, ApoE, SNPs ◦ E.g Telomeres DNA damage and repair analysis Methodological validation: Protein assays ◦ Detection limits ◦ Serum/plasma/blood cells/culture supernatants ◦ Coefficients of variation ◦ Sandwich ELISA, Competitive ELISA ◦ Dilutions ◦ 96-well and 384-well plates ◦ Sensitivity ◦ Oxidative Damage ◦ Enzymatic activity (e.g. telomerase)
In-house Measurements ◦ Handling of complex sample logistics, regulatory frameworks ◦ Rational use of samples, maximum potential ◦ Large collections: traceability and timely delivery of results ◦ Beyond routine Clinical Biochemistry – added value ◦ “Made to measure”- with you through the whole process ◦ Explore new methodologies ◦ Detailed costing
Case Study 1 – StemBANCC StemBANCC 5 year programme (EU 7th FP) CARU team part of the multi-centre recruiting process with blood samples requiring a standardized processing for biobanking by a third party. Biomarkers lab ◦ Coordinating with Biobank lab ◦ Coordinating with CARU nurses ◦ On the day CARU nurse assesses participant, collect blood and within 5min the sample is at Biomarkers lab for • Sample processing • Cryopreservation • Safe storage and final shipping to Biobank
Case Study 2 – Starlings Biomarker lab previous work in humans: potential to translate onto birds Telomeres ◦ Develop and improve a previous method on bird telomeres ◦ Isolate DNA and assess telomeres at large scale (N~600) Inflammatory markers in birds ◦ Develop and validate method for IL-6 and hsCRP in birds with very limited amount of plasma. ◦ Measure IL-6 and hsCRP on same sample (N>120 samples) ◦ Final publication: from writing up methods to replies to reviewers Genotyping for sexing of birds ◦ Test 3 previous methods on genomic sexing of these birds ◦ Evaluate gender on large collection of samples (N~400)
Case Study 3 - Pre-eclampsia RVI Biomarker lab previous work on inflammatory markers: Apply those biomarkers and develop new ones (Myeloperoxidase, MPO) in Pre-eclampsia. ◦ Assess IL-6 and TNA-alpha in plasma samples (N=190) ◦ Test 3 possible samples for measurement of Myeloperoxidase ◦ Develop, up-scale and increase sensitivity of current MPO assay ◦ Higher throughput and Higher reproducibility
General Support Ad hoc technical support ◦ When you needed, where you needed Support with Costings and Grant applications ◦ Flexible detailed costings ◦ Co-applicants or service providers Post-graduate training and support Writing up: Methods, reviewers reply, etc.
For further details, please contact: Dr Carmen Martin-Ruiz Biomarkers Lab Manager Campus for Ageing and Vitality Newcastle upon Tyne, NE4 5PL Tel: +44(0)191 2081107 E-mail: carmen.martin-ruiz@ncl.ac.uk http://www.ncl.ac.uk/ion/about/facilities/biomarkers/
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