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Mr Michael O'Sullivan,Assistant Director for Buildings & Technical Services,UCD Conway Institute of Biomolecular & Biomedical Research,University College Dublin,Belfield, Dublin 4
T: (+353 1) 716 6705E: [email protected] Website: www.ucd.ie/conway/research_technologies.html
Funded through the Programme for Research in Third Level Institutions,administered by the Higher Education Authority
We gratefully acknowledge the contributions made by statutory, charitable andphilanthropic organisations to facilitate the work of UCD Conway Institute ofBiomolecular & Biomedical Research.
UCD Conway Institute of Biomolecular & Biomedical Research
CORE TECHNOLOGIES
CORE TECHNOLOGY
1.
To date, UCD Conway Institute has invested
several million euros in the development of its
core technology programme. This programme
consists of a number of facilities where
equipment is centrally located, properly
maintained and managed by a cohort of technical
staff who provide technical support to Conway
researchers. Training is provided in sample
preparation, instrument use and, depending on
the technology, subsequent analysis of raw data.
This allows researchers to access high value
specialised equipment, which is properly
maintained and managed.
CORE TECHNOLOGY
2.
Affymetrix GeneChip facility
Applications:
The Affymetrix GeneChip technology platform consists of high-densitymicroarrays and tools to help process and analyse those arrays, includingstandardized assays and reagents, instrumentation, and data managementand analysis tools. GeneChip microarrays consist of small DNA fragmentsknown as probes, chemically synthesized at specific locations on a coatedquartz surface. The precise location where each probe is synthesized iscalled a feature, and millions of features can be contained on one array. By extracting and labelling nucleic acids from experimental samples, andthen hybridising those prepared samples to the array, the amount of labelcan be monitored at each feature, enabling a wide range of applications on awhole-genome scale - including gene- and exon-level expression analysis,novel transcript discovery, genotyping, and resequencing. Microarrayanalysis can also be combined with chromatin immunoprecipitation toperform genome-wide identification of transcription factors and theirrespective binding sites.
Instrumentation: • GeneChip 3000 7G scanner• Automated Fluidics 450 station• Hybridisation oven• GeneChip workstation with AGCC Software • Agilent Bioanalyser• Nanodrop Spectrophotometer
Services:
Contact: Ms Alison Murphy
T: (+353-1) 716 6955
• Initial meeting to discuss experimentaldesign and strategy
• Advice on sample preparation andprovision of protocols
• Optional sample preparation and labelling service
• Quality assessment of total RNA and cRNAusing the Agilent Bioanalyser
• Array hybridisation, washing, staining andscanning
• Assessment and monitoring of arrayquality
• Primary data analysis with AGCC Software and provision of QC report and all data files
Real Time PCR Facility
Applications:
Real-Time PCR is a highly specific and sensitive method of DNA and RNAquantification. It allows the monitoring of PCR amplification andcharacterisation of samples at the point of initial detection of PCR productrather than the amount of product accumulated after a fixed number ofcycles. Both absolute and relative quantification studies can be carried outand applications include viral load, gene copy number and relative geneexpression determination. Real-Time PCR instrumentation may also be usedfor allelic discrimination assays.
Instrumentation: Two ABI 7900HT Sequence Detection Systems are in operation in this facility. These instrumentssupport both Taqman and Sybr Green chemistries and comprise:
• Standard and Fast 96 and 384 well thermal cycling blocks
• Laser and CCD camera detector
• Sequence Detection Software v 2.2.1
• Primer Express Software v 2.0
• Low Density Array system
Service: The Real-Time PCR core facility provides a range of technical services and expertise in this area toresearchers, which include:
• Advice on experimental design
• Protocols
• Assay design & ordering
• Training
• Sample running service
• Results analysis
Contact: Dr Giuliano Elia (Director)/ Ms Catherine Moss
T: (+353-1) 716 6946
E: [email protected] / [email protected]
CORE TECHNOLOGY
3.
CORE TECHNOLOGY
4.
Flow Cytometry Facility
Applications:
Flow cytometry can be used to measure intrinsic (size, shape, granularity etc.) and extrinsic cell characteristics (DNA, internal andexternal receptors etc.). This has many applications in humans, animals,plants and microorganisms. The most common applications are cell cycle,apoptosis/necrosis, ploidy determination, immunophenotyping, proteinexpression, Ca2+ concentration, etc.). Cell sorting allows the physicalisolation of cell populations for further procedures such as cell culture and studies of protein expression.
Instrumentation: COULTER EPICS XL-MCL Simultaneous measure of FSC, SSC and up to 4 colours using a 488nm laser and has an automatedsample loader, which permits a throughput of 100 samples/hour.
COULTER FC500 This equipment, the next generation of the Epics XL-MCL, is able to analyse up to 5 colours using a488nm laser. It has an automated sample loader.
DAKO CYAN™-ADP We have two Dako Cyan ADP, each with three lasers: one with 351nm (UV), 488nm (blue) and635nm (red) and the other one with 405 nm (UV), 488nm (blue) and 635nm (red). This allows usto detect up to 11 parameters at the same time, this offers complete compensation and analysisrates of up to 50,000 events/second.
BD FACSARIA CELL SORTER High-speed cell sorter (40,000 events/second) with 3 lasers: 488nm (blue), 633nm (red) and407nm (violet) for detection of FSC, SSC and up to 11 parameters. It is able to sort 4 populations ofcells at the same time and perform cell cloning in well plate platforms with a 100% purity level.
Service:The flow cytometry facility provides a range of services including training, instrumentation,workstation, software for analyses and technical expertise. Trained users prepare and run their ownsamples. Untrained users may avail of the sample running service and results analyses.
Contact:Dr. Alfonso Blanco
T: (+353-1) 716 6836 / 6947
Confocal Microscopy & Digital Imaging Facility
Applications:Confocal microscopy facilitates broad spectra of techniques for the detection, spectral separationand visualisation of single and multiple fluorescent labelled samples including fluorphores withoverlapping spectra. The system also facilitates other techniques such as FRET, FRAP and ionimaging. Designated confocal systems facilitate electrophysiology/patch-clamping techniques andintravital microscopy allowing the visualisation of complex reactions between flowing blood cellsand blood vessel walls in living organisms.
Live cell imaging facilitates the visualisation of live cells using a motorised inverted microscope fortransmitted light, brightfield, phase contrast, DIC and fluorescence microscopy. System featuresinclude time-lapse, multi-channel, mark and find, extended depth of focus and auto focus as well astemperature, CO2 and O2 control. The system also encompasses FRET analysis.
PALM® MicroBeam and Laser system facilitates precise non-contact laser basedmicromanipulation and permits any combination of cutting and catapulting the sample, with orwithout the use of PALM® Membrane slides for collecting morphologically intact cell areas.Protocols for DNA, RNA, protein, microarray and mass spectrometric analysis can be appliedwithout difficulty.
Instrumentation: • Zeiss LSM 510 UVMETA confocal microscope system with an interchangeable Axiovert 200M
and Axioskop 2 FSMOT microscope
• Two Zeiss LSM5 PASCAL systems for physiology and intravital research
• Zeiss Live Cell Imaging system based on the Axiovert 200M microscope
• PALM® MicroBeam and laser micromanipulation system
Service:The facility provides a range of services including training in instrumentation, technical expertiseand software for post-image acquisition analysis. Assisted sessions are provided on request.
Contact:Ms Ann Cullen
T: (+353-1) 716 6741
CORE TECHNOLOGY
5.
CORE TECHNOLOGY
6.
Histology Facility
Applications:
The facility provides for all aspects of TMA technology from tissueprocessing to TMA construction to automated immunohistochemistry. Usingthe Aperio CS scanner, capture of digital histopathological images and theautomated quantitative assessment of nuclear, membranous andcytoplasmic staining on both TMAs and full face sections is possible.
Instrumentation: • Leica histokinette processor
• Leica EG1150H
• Several microtomes
• Automatic Stainer Leica XL
• Automatic coverslipper LeicaCV5030
• Labvision pre-treatment (PT) module
• Automatic Immunostainer (Lab Vision 360)
• Manual tissue microarrayer
• Aperio CS scanner
• Multiheaded microscope
Service: • Processing, paraffin embedding, sectioning and production of slides for staining
• Routine Haematoxylin and Eosin staining with facilities to programme for special stains
• Construction and sectioning of TMAs using the Beecher Manual Tissue arrayer
• Various antigen retrieval methods
• Instruction in all aspects listed above
• Automated immunohistochemical optimisation and staining using the Lab Vision 360 instrumentation
• Automated coverslipping using the Leica CV5030 instrumentation
Contact:Mr George Keating
T: (+353-1) 716 6741
Electron Microscopy Facility
Applications:
The facility allows comprehensive ultrastructural research to be undertakenin both the material and biological sciences. Applications range fromultrathin section analysis of cells and tissues with detail maximally resolvedat a few nanometres to topographical studies using scanning electronmicroscopy and elemental analysis.
Instrumentation: • 3 high-resolution transmission electron microscopes (TEM)
• 1 scanning electron microscope (SEM)
• 1 cryo-stage SEM equipped with a modern elemental analysis system
• 4 ultramicrotomes and 1 cryo-ultramicrotome
• Wide range of SEM ancillary preparation instruments
• Light microscopy analysis is also well supported
Service:In addition to providing a comprehensive electron microscopy facility for the use of all staff in UCD,services include:
• Technical expertise to assist in the widest possible range of applications of electron microscopy
• Development of new or existing applications of electron microscopy to their research
• Comprehensive training allowing users to become proficient users of electron microscopes
• High quality electron microscopy consultancy service for external agencies
Contact:Dr David Cottell
T: (+353-1) 716 7133
CORE TECHNOLOGY
7.
CORE TECHNOLOGY
8.
Proteome Research Centre
Applications:
The PRC applies leading-edge proteomic technologies to identify diagnosticand prognostic biomarkers of diseases and potential targets for therapeuticintervention in human disease. The PRC, created in 2003, builds on thecompetence in the proteomic field of worldwide recognized leaders likeSteve Pennington and Mike Dunn.
Instrumentation:The Mass Spectrometry resource is a facility dedicated to biological mass spectrometry. It avails ofstate-of-the-art mass spectrometry instrumentation for a wide range of proteomic applications.The 4800 MALDI-Tof / Tof mass spectrometer with autoloader for high throughput MALDI-MS /MS is applied to gel-based and off-line liquid chromatography (LC) MS/ MS workflows. Two linearion trap electrospray mass spectrometers, a linear ion trap equipped with Electron TransferDissociation source, an Orbitrap, a quadrupole time-of-flight, a hybrid linear ion trap-triplequadrupole and a linear ion trap electrospray mass spectrometer coupled to a Fourier TransformIon Cyclotron Resonance detector cover all possible needs in liquid-chromatography-tandem massspectrometry. The technological offer of the Mass Spectrometry Resource is completed by roboticstations for gel spot picking and in-gel tryptic digestions.
The Separations laboratory is a facility dedicated to electrophoretic gel separation of proteins prior to their identification by mass spectrometry. It avails of instrumentation for 1-D gelelectrophoresis, 2-D gel electrophoresis (2-DE), image scanners including a three colour laserfluorescence scanner (Typhoon) for three colour difference in gel electrophoresis (DIGE) 2-DE. LC-based workflows are supported by the multi-dimensional LC system and a nano-LC system andare coupled to a MALDI target loading robot to support off-line LC MALDI-MS.
These facilities are used to support the research projects of a vast research community mostlybased in Conway Institute and other UCD Schools, but are also open to other academic institutionsand commercial companies worldwide.
Contact: Dr. Giuliano Elia (MSR Director), Ms Ashling Gantly (secretary), or Ms Caitriona Scaife
T: (+353-1) 716 6986/6714 F: (+353-1) 716 6703
Proteinchip Facility
Applications:
This technology utilises surface enhanced laser desorption/ionisation(SELDI) technology, in combination with time-of-flight mass spectrometry(ToF-MS), to facilitate protein profiling of complex biological and clinicalsamples. Offering a rapid and reproducible method to analyse proteins fromcrude biological samples, SELDI facilitates transition from discovery toassay on a single platform.
Instrumentation: Equipped with Ciphergen’s SELDI ProteinChip system, a powerful platform for the rapid discoveryand assaying of multiple biomarkers associated with various diseases and other pathological states.
Service:The ProteinChip facility allows users to process up to 1,000 samples per day on our recentlyupdated Enterprise Series 4000 system, and is fully accessible on a cost-recovery basis. Both in-house and external training is available on the instrument and associated software.
Contact: Professor William Gallagher or Dr Linda Whelan
T: (+353-1) 716 6743 F: (+353-1) 283 7211
E: [email protected] / [email protected]
Funding for the ProteinChip Facility was provided by the Health Research Board under itsEquipment Programme scheme, along with additional support from Science Foundation Irelandand UCD Seed Funding.
CORE TECHNOLOGY
9.
CORE TECHNOLOGY
10.
DNA Sequencing and Oligonucleotide Ordering Service
Applications:
DNA sequencing and oligonucleotide synthesis are essential tools used by most scientific investigators at some stage during their research. To aid this activity the technical aspect of this service was out-sourced to a genomics company. There are a broad variety of uses for the productsprovided. In general, the templates used for sequencing are either plasmidsor PCR products. However, large library screens or primer walking of largeconstructs can also be carried out. The synthesised oligonucleotides arepurified appropriately and used for conventional or real-time quantitativePCR etc. siRNA duplexes can be used to silence the genes of interest andexamine their expression profile.
Instrumentation: This facility is predominantly web-based. There are many facets:
• Online ordering system
• Convenient barcode labelling system for sequencing
• Free courier service for the collection of sequencing samples
• Sequencing results are downloadable from the website in various formats; text, .pdf and .scf files
• Free courier service for delivery of synthesised oligonucleotides
• Orders can be tracked online through the website
Service: The service is co-ordinated and managed through the Transcriptomics Core Facility. Administrationof the service includes contract negotiation, provision of purchase order numbers, facilitatingdeliveries and courier services and processing company invoices. Advice is available on all aspectsof the service, from sample preparation and design to utilising the online ordering system.
Contact: Ms Janet McCormack
T: (+353-1) 716 6956
CORE TECHNOLOGY
11.
Digital Slide Scanning and Image Analysis Facility
Applications:
The technology is a microscope slide scanner, which digitises entire slidesat high quality resolution, whether they are full-face sections, cell pelletarrays or tissue microarrays. The system comes with a host of softwareapplications that enable you to scan, compress, view and analyse yourdigital slides. This powerful system allows you to experience fast, high-capacity digital scanning. The digital slides can be managed through a comprehensive, web-based digital pathology information managementsoftware suite. This was developed for digital slide viewing andconferencing, data archival, intelligent retrieval and image analysis. Its high-throughput of superior images renders its application fundamentalin drug discovery, cancer research and telepathology to name just a few.
Instrumentation:• Aperio ScanScope© XT system with 120-slide capacity
• ImageScope" software, allows viewing and sharing of slides. Provides features such as: instant zoom and pan of images, annotation or analysis of digital sides.
• Access to various algorithms for image analysis of immunohistochemical staining etc., in conjunction with ImageScope"
• Spectrum”, a web-based software that facilitates the management of digital slides
• Digital Slide Studio software is an easy to use tool for performing simple image processing functions
Instrumentation:The Digital Slide Scanning and Image Analysis Facility provides a range of services and technicalexpertise in this field to researchers including:
• Advice on slide preparation for the purposes of scanning
• Training on all aspects of using the Aperio ScanScope© system and data management
• Guidance with regard to scanning a range of different slides
• Fully trained users have full access to the facility
• Assisted sessions provided on request
Contact: Ms Janet McCormack
T: (+353-1) 716 6956
NOTES
12.
