The Bio-Screening Core Facility PETER BANKS PETER.BANKS@NCL.AC.UK - - PowerPoint PPT Presentation

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The Bio-Screening Core Facility PETER BANKS PETER.BANKS@NCL.AC.UK - - PowerPoint PPT Presentation

Aug 28, 2023 10 likes •194 views

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

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SLIDE 1

The Bio-Screening Core Facility

PETER BANKS PETER.BANKS@NCL.AC.UK CARMEN MARTIN-RUIZ CARMEN.MARTIN-RUIZ@NCL.AC.UK

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SLIDE 2

Bio-Screening Facility Information

High Throughput Screening Facility Peter Banks

Peter.Banks@ncl.ac.uk

Cookson/Leech Building Medical School https://www.ncl.ac.uk/htsf/ Biomarker Facility Carmen Martin-Ruiz

Carmen.Martin-Ruiz@ncl.ac.uk

Claire Kolenda

Claire.Kolenda@ncl.ac.uk

Craig Parker

Craig.Parker@ncl.ac.uk

Ageing Research Labs Campus for Ageing and Vitality https://www.ncl.ac.uk/ion/about/facilities/biomarkers/

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SLIDE 3

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
  • f organisms
  • Drug screening in collaboration with

LifeArc

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SLIDE 4

Equipment

2 sites – 2nd floor Cookson and 2nd 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
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SLIDE 5

Genetic Screening

Cell based genetic screening is the combination of laboratory automation with high throughput biological reagents.

  • Identify new research directions
  • Preliminary data generation for grant applications

High throughput reagents are specifically designed for automation

  • Excellent coverage for large scale experiments
  • Post screen validation is critical to success

Yeast collections

  • Deletion library ~4500 strains
  • Overexpression library ~6000 strains
  • DaMP Library ~800 strains
  • Histone point mutant library ~ 400 strains
  • Temperature sensitive collections

Bacterial collections

  • Bacillus subtilis
  • Escherichia coli

Dharmacon Human siRNA library

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SLIDE 6

Dharmacon siRNA Library

Dharmacon siGENOME SMARTpool siRNA library

  • siGENOME – type of siRNA
  • 4 siRNAs to a single gene in one well – SMARTpool
  • ~7500 genes over 28 plates
  • 1nmol in four separate 384 well plates
  • Screen 400-2000 plates
  • Supply the gene list
  • Add individual controls to experiments

Cherry pick individual 384 or 96 well plates

  • G Protein Coupled Receptors
  • Protein Kinases -3 plates
  • Ion Channels
  • Phosphatases – 1 plate
  • Proteases
  • Ubiquitin Conjugation 1 - Cullins, E1, E2, HECT E3

Ligases

  • Ubiquitin Conjugation 2 - F-box, SOCS box E3 Ligases
  • Ubiquitin Conjugation 3 - RING finger and RING

finger-like E3 Ligases

  • Drug Targets
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SLIDE 7

Drug Screening

The HTSF has a collaboration with the life science charity LifeArc to provide FMS researchers with drug screening

  • LifeArc links basic research to industrial collaborators
  • Expertise – medicinal chemistry, IP and screening
  • Provided a 20,000 compound library to screen here

Free to access the libraries

  • LifeArc need to approve the screen

Libraries supplied blind

  • Results must be fed back to LifeArc to deconvolute

20ul of drug at 10mM in DMSO

  • 50-200 plates

Index library

  • ~12,000 compounds representative of full LifeArc

collection

Kinase library

  • ~6,700 compounds predicted to inhibit kinases

Natural product library

  • ~1000 purified novel natural products from plants or

fungi

FDA approved drugs library

  • ~1,000 compounds
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SLIDE 8

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
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SLIDE 9

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

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SLIDE 10

Sample Handling & Technical Support

Blood processing and blood cell separation

  • PBMC isolation, buffy coats,
  • PAXgene tubes
  • Cell Preparation Tubes (CPT)
  • Red cells conditioning

Plasma and Serum

  • Pre-treatment and conditioning (anti-oxidants)
  • Aliquoting

Coordination with Clinical/Research Teams

  • RVI/Freeman Labs and CARU
  • Nationwide and overseas

Sampling logistics

  • Optimal use of blood samples.
  • Suitability of anticoagulant (or not!)
  • Phlebotomy requirements: blood volumes, timings,

priorities

Sample tracking

  • Labelling and Barcoding
  • Achiever compatible datasets
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SLIDE 11

Follow-up Sample Processing

DNA Isolation

  • Phenol/Chloroform or Spin columns
  • Blood samples
  • Tissues
  • Cell pellets
  • Saliva swabs
  • Blood spot cards
  • Feathers
  • Nails

DNA & RNA isolation

  • From same sample
  • Fresh bloods, tissue
  • Cell pellets

RNA Isolation

  • PAXgene tubes
  • Fresh bloods
  • Cell pellets

Specialized processing

  • Cryogenic conditioning of cells, cryopreservation
  • Cell culture
  • Cytokines: LPS-stimulation, blood cultures
  • Flow cytometry

.... and more

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SLIDE 12

Biomarker Analysis

RNA and Gene expression analysis

  • RT-qPCR, 384-well capacity,
  • TaqMan Gene expression assays,
  • TaqMan MicroRNA assays

DNA and Genotyping

  • Genotyping by Agarose Gels (Mice Ear Notches)
  • Genotyping by qPCR (inc.TaqMan™ Genotyping)
  • Telomeres, ApoE, SNPs

DNA damage and repair analysis Protein assays

  • Serum/plasma/blood cells/culture supernatants
  • Sandwich ELISA, Competitive ELISA
  • 96-well and 384-well plates
  • Oxidative Damage
  • Enzymatic activity (e.g. telomerase)

Novel biomarkers:

  • From cell culture to humans

Novel sources of material:

  • From humans to animals

Epidemiological scaling:

  • From single sample to 384-well plates
  • E.g Telomeres

Methodological validation:

  • Detection limits
  • Coefficients of variation
  • Dilutions
  • Sensitivity
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SLIDE 13

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
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SLIDE 14

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
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SLIDE 15

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)
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SLIDE 16

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
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SLIDE 17

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.

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SLIDE 18

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/