Techniques and Strategies 2019 – Abstracts & Useful Literature


Next-generation Sequencing
Dr Lisa Mc Dermott (TCD)

Next-generation sequencing (NGS) is technology that can generate DNA sequence data in a manner that is fast and accurate. This talk will provide an introduction to Next-Generation Sequencing technology and its many applications.

Suggested reading

Epigenetics: What is it and why is it important?
Prof Gerard Brien (TCD)

Studying the epigenetic regulation of DNA-templated processes is a highly active area of biomedical research, with significant implications for our understanding of basic biological processes and disease states. The term epigenetics was originally coined to describe heritable changes in cellular phenotypes that were independent of alterations in DNA sequence. Today, epigenetics is most commonly used to describe chromatin based events that regulate patterns of gene expression. The mechanisms involved primarily relate to the covalent modification of the DNA and histone proteins that make up the chromatin fibre. These modifications effect chromatin structure and function by altering accessibility of the genetic information contained within our genomes. Significantly, in recent years we have realised that these mechanisms are frequently disrupted in human disease; particularly in cancer. Importantly, research in this area is providing insights into the pathological molecular mechanisms at the heart of disease development.

Suggested reading

Epigenetics by C. David Allis, Thomas Jenuwein and Danny Reinberg
CHAPTER 3: Overview and Concepts
CHAPTER 10: Chromatin Modifications and Their Mechanism of Action

Chromatin modifications and their function Cell. 2007 Feb 23;128(4):693-705. Kouzarides T

Mass Spectrometry in Biological and Medical Research

Prof Matthias Wilm (UCD)

With the discovery of the two soft ionisation methods electrospray and matrix assisted laser desorption/ionization mass spectrometry as an analytical technique made its way into biological research. Today this technique is the major tool for protein identification, the characterisation of secondary modifications and the analysis of lipids and small molecules in biological research.

With thousands of proteins identified in each analysis the information of how much of each protein was in the sample became of outermost importance. With new acquisition regimes and software tools the ability to quantify proteins in a reliable and reproducible way just got massively improved. By this mass spectrometry based protein characterisation will be broadly applicable to medical research.

Useful Literature

The Coming Age of Complete, Accurate, and Ubiquitous Proteomes
Matthias Mann,1 ,2 , Nils A. Kulak,1 Nagarjuna Nagaraj,1 and Ju¨ rgen Cox1
1Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
2The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen,
2200 Copenhagen, Denmark

Multiplexed peptide analysis using data-independent acquisition and Skyline
Jarrett D Egertson1, Brendan MacLean1, Richard Johnson1, Yue Xuan2 & Michael J MacCoss1
1Department of Genome Sciences, University of Washington, Seattle, Washington, USA. 2Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany. Correspondence
should be addressed to M.J.M. (
Published online 21 May 2015; doi:10.1038/nprot.2015.055

Stem Cells – Use, Isolation and Analysis

Dr Maojia Xu (NUI Galway)

Stem cells are relatively unspecialised cells lacking tissue-specific characteristics. Under appropriate conditions they can generate one or multiple specialised cell types in a process called ‘differentiation’. Stem cells show enormous therapeutic potential based on their ability to generate cells for repair or regeneration of damaged tissues and organs. Stem cells can also be used as a source of ‘normal’ human cells to study processes such as development and wound healing as well as investigating human-specific toxicity testing of drugs.
This lecture will describe stem cells sources, characteristics and analysis. Hopefully it will give you some ideas about how stem cells could contribute to your research.

Useful Literature

Modeling Development and Disease with Organoids
Hans Clevers1,
1Hubrecht Institute/Royal Netherlands Academy of Arts and Sciences, Princess Maxima Centre and University Medical Centre Utrecht,
3584CT Utrecht, The Netherlands

The Molecular and Cellular Choreography of Appendage Regeneration
Elly M. Tanaka1,
1DFG Research Center for Regenerative Therapies, Technische Universita¨ t Dresden Fetscherstrasse 105, 01307 Dresden, GERMANY

Stem Cells: A Renaissance in Human Biology Research
Jun Wu1,2 and Juan Carlos Izpisua Belmonte1,
1Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA
2Universidad Cato´ lica San Antonio de Murcia (UCAM) Campus de los Jero´ nimos, 135, Guadalupe 30107, Murcia, Spain

Pluripotent stem cells in disease modelling and drug discovery.
Avior Y1, Sagi I1, Benvenisty N1.
Nat Rev Mol Cell Biol. 2016 Mar;17(3):170-82. doi: 10.1038/nrm.2015.27. Epub 2016 Jan 28.

Manipulating the Rodent Genome Using Transgenic and Gene Targeting Techniques

Dr Tom Moore (UCC)

Manipulation of gene expression and function in living rodents is virtually a prerequisite for studying developmental genetics and pathology and the creation of rational models of human genetic diseases. However, the available techniques remain challenging and expensive. I will provide an overview of the underlying principles and practical applications of these techniques, which are usually applied to mice, but with increasing frequency to rats and, to a lesser extent, other mammalian species. Techniques covered in this lecture will include electroporation, oocyte microinjection and lentivirus mediated transgenesis, gene targeting in embryonic stem cells, gene trap libraries, inducible vectors, shRNA, zinc finger nucleases, TALENs, and CRISPR. I will provide an overview of national and international centres engaged in large-scale production of mutants and suggestions for additional reading.

Useful Literature

Selected publications

Kumar et al. 2009; doi:10.1007/978-1-60327-378-7_22
Schaefer et al. 2017; doi:10.1038/nmeth.4293
Cohen 2016; doi:10.1126/science.aal0334
Quadros et al. 2017; DOI 10.1186/s13059-017-1220-4

Some service centres and companies

Protein Expression and Purification
Dr Henry Windle (TCD)

There is a growing need to generate and purify multiple types of recombinant proteins for use in diverse areas such as structural studies, diagnostic/pharmaceutical use, biochemical studies, industrial use and for use as research reagents and antibody production. It is relatively easy to clone a gene and express the gene product in a suitable host by making use of the many commercially available versatile expression systems and hosts. Problems are generally encountered at the protein purification stage however. This presentation will cover the basics of protein expression and purification. Emphasis will be placed on alternative strategies and issues that should be considered prior to selection of specific expression systems and purification strategies.

Useful literature

Methods in Enzymology 559, 2-148 (2015): Laboratory methods in Enzymology: Protein part D – This collection represents a very comprehensive overview of protein expression and purification strategies
Burgess R. (2012) Fusion tags: A collection of papers. Protein Express. Purif. 81.
GE Healthcare Life Sciences Handbook Collection: ‘Recombinant Protein Purification: principles and Methods.

Immunodetection Methods on Cell and Tissue Extracts

Dr Ann Hopkins (RCSI)

Antibodies have been used as tools to detect and characterise proteins for decades. With the ongoing development of high throughput protein detection techniques such as tissue microarrays and reverse phase protein arrays, a real challenge now exists to contextualise the cellular locations, levels of expression and the functions of identified proteins. In this lecture, the fundamental immunological principles underlying protein detection in cells and tissues will first be outlined. This will set the scene for discussing experimental approaches to detect either protein expression levels, protein localization or protein-protein interactions. Accordingly the applications of immunodetection in a modern molecular context will be illustrated, including western blotting, ELISA, immunohistochemistry, immunofluorescence, tissue microarrays, co-immunprecipitation, electromobility shift assays, chromatin immunoprecipitation (ChIP) and antibody arrays. Common experimental pitfalls and interpretational challenges will be discussed. After this lecture, the participants should have an appreciation of how to design and optimise experiments to detect proteins in a variety of contexts and models.

Useful Literature

Protein Microarrays for Personalized Medicine. Xiaobo Yu, Nicole Schneiderhan-Marra, Thomas O. Joos
DOI: 10.1373/clinchem.2009.137158 Published February 2010

Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients.
Alicia D. Powers and Sean P. Palecek. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, USA. Journal of Healthcare Engineering. Volume 3 (2012), Issue 4, Pages 503-534.

Protein expression profiling arrays: tools for the multiplexed high-throughput analysis of proteins. Jens R Sydor and Steffen NockEmail author. Proteome Science20031:3.



Fluorescence microscopy based high-content screening: Turning pixels into quantitative data
Dr Eugene Dempsey (UCD)

High-content screening (HCS) combines automated microscopy and computer based image analysis to generate quantitative data which can be used to accurately describe phenotypic changes in cells. HCS typically utilises fluorescence based microscopy, thereby, allowing multiple markers of different cellular components to be imaged in parallel. Quantitative data on each cellular component is then extracted using sophisticated image analysis pipelines for further downstream analysis. To date, HCS has been successfully employed in a wide number of fields from novel drug discovery and toxicology to the mapping of biological pathways. This seminar will aim to provide an overview of fluorescence based microscopy, current HCS technology and typical HCS workflows. Example data will be used to demonstrate the power of combining RNA interference with HCS. Finally, looking to the state of the art, we will discuss 3-dimensional in vitro models and HCS.

1. Bray MA, Carpenter A. Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
2. Martin S, Buehler G, Ang KL, Feroze F, Ganji G, Li Y. Cell-Based RNAi Assay Development for HTS. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
3. Buchser W, Collins M, Garyantes T, Guha R, Haney S, Lemmon V, et al. Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
4. Strovel J, Sittampalam S, Coussens NP, Hughes M, Inglese J, Kurtz A, et al. Early Drug Discovery and Development Guidelines: For Academic Researchers, Collaborators, and Start-up Companies. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
5. Boutros M, Heigwer F, Laufer C. Microscopy-Based High-Content Screening. Cell. 2015;163(6):1314-25.

Metabolomics and examples of use
Prof Lorrainne Brennan (UCD)

Metabolomics is the study of small molecules called metabolites which can reveal information on metabolic pathways and their alterations under different conditions. This lecture will cover the basics of metabolomics and highlight key examples of how it can be used.


  1. Wishart, D. S., Emerging applications of metabolomics in drug discovery and precision medicine. Nat Rev Drug Discov 2016, 15, 473-84.
  2. Liggi, S.; Griffin, J. L., Metabolomics applied to diabetes-lessons from human population studies. The international journal of biochemistry & cell biology 2017, 93, 136-147.

CRISPR-What’s the fuss about?

Prof. Vincent P. Kelly (TCD)

The tools for programmable engineering of the genome have been evolving rapidly; zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently CRISPR-Cas–mediated gene targeting. CRISPR in particualar is being singled out for its ease of use. These technologies are having a huge impact on the type of questions that can be adressed in basic research and opening up unprecedented approaches to the treatment of numerous monogenetis diseases in humans. In its simplest sense the programmale nucleases induce site-specific DNA cleavage in the genome, that are repaired through endogenous mechanisms and allow high-precision editing of point mutations, reporter casettes, conditional allele etc. There is little doubt that this technology will have far ranging conseqences in bioloigcal and medical science into the future.


Gaj, T., Gersbach, C.A. and Barbas III, C.F., 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology, 31(7), pp.397-405.

Bak, R.O., Gomez-Ospina, N. and Porteus, M.H., 2018. Gene editing on center stage. Trends in Genetics.

Crystal Digital PCR™: how partition-based PCR continues to revolutionize nucleic acid detection

Emma Sharkey, (Stilla Technologies)

The commercialization of digital PCR platforms has sparked a revolution in quantitative nucleic acid detection over the past decade. As a result of its high accuracy, sensitivity and robustness, digital PCR has proven reliable in a multitude of applications, ranging from rare event detection, NGS library quantification, rapid assessment of genome editing events and therapeutic disease monitoring from liquid biopsy and tissue samples.

In this seminar, we will give insights into this cutting-edge technology and present some of the current and future developments in the field of digital PCR using the Naica™ System, a unique 3-color detection digital PCR platform.



Model Organisms
Dr Stephen Carter (UCD)

The aim of this lecture is to give an overview of the advantages and disadvantages of various leading model organisms which are routinely used in biomedical research. We will discuss the use of invertebrate and vertebrate models and their application to the study of development and disease research. We will cover transgenic, mutagenic and gene-edited models of disease using both targeted methods of genetic manipulation and random screens. We will also cover chemical screening methods in animal models. These methods have greatly improved our understanding of biology and underlying gene function. The various models used in vision research will be used as case studies.

Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research:

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases:

Translational fMRI Functional Connectomics
Dr Clare Kelly (TCD)

Task-independent or “resting state” functional magnetic resonance imaging (fMRI) approaches (“functional connectomics”) have revolutionized our understanding of brain functional organisation and have driven significant advances toward the goal of identifying valid and reliable biomarkers of psychiatric illness. Functional connectomics offers the promise of a truly translational tool; the correspondence between functional circuits identified in the human, macaque, and rodent has been demonstrated. This talk will provide an overview of fMRI-based functional connectomics and will illustrate how translational work has the potential to provide mechanistic insights into how disturbances in typical brain development give rise to psychiatric and neurological disorders.

Useful Literature

Grandjean, J., Canella, C., Anckaerts, C., Ayrancı, G., Bougacha, S., Bienert, T., Buehlmann, D., Coletta, L., Gallino, D., Gass, N. and Garin, C.M., 2019. Common functional networks in the mouse brain revealed by multi-centre resting-state fMRI analysis. NeuroImage, p.116278.

Stevens, H. E., & Vaccarino, F. M. (2015). How animal models inform child and adolescent psychiatry. Journal of the American Academy of Child and Adolescent Psychiatry, 54(5), 352–359.

Di Martino, A., Fair, D. A., Kelly, C., Satterthwaite, T. D., Castellanos, F. X., Thomason, M. E., et al. (2014). Unraveling the miswired connectome: a developmental perspective. Neuron83(6), 1335–1353.

Gorges, M., Roselli, F., Müller, H.-P., Ludolph, A. C., Rasche, V., & Kassubek, J. (2017). Functional Connectivity Mapping in the Animal Model: Principles and Applications of Resting-State fMRI. Frontiers in Neurology8, 200.

Hutchison, R. M., & Everling, S. (2012). Monkey in the middle: why non-human primates are needed to bridge the gap in resting-state investigations. Frontiers in Neuroanatomy6, 29.



Flow Cytometry, Cell Sorting and Flow Imaging
Dr Alfonso Blanco (UCD)

Flow cytometry is a method for qualitative and quantitative analysis of components or structural features of cells, primarily by optical means, but also particles. Although it makes measurements on one cell at a time, it can process thousands of cells per second. Since cell types can be distinguished by quantitating structural and/or physiological features, flow cytometry can be used to count prokaryotic or eukaryotic cells of different types in complex mixtures. Flow cytometry PDF

Useful Literature

Flow Cytometry – A Basic Introduction (by Mike Ormerod)

Clinical Flow Wiki

Compensation by Mario Roederer

How to perform cell counts using a hemocytometer

ThermoFisher. Resource Center. Introduction to Flow Cytometry. Analyzing Flow Cytometry Data

ThermoFisher. Fluorescence Tutorials

ExCyte. Expert Cytometry

Purdue University Cytometry Lab

BiteSized Immunology

Imaging Using Fluorescent/Confocal Microscopy
Dr Gavin McManus (TCD)

Fluorescence microscopy is an important and fundamental tool for biomedical research. Optical microscopy is almost non-invasive and allows highly spatially resolved images of organisms, cells, macromolecular complexes and biomolecules to be obtained. Generally speaking, the architecture of the observed structures is not significantly modified and the environmental conditions can be kept very close to physiological reality. The development of fluorescence microscopy was revolutionised with the invention of Laser Scanning Confocal Microscopy (LSCM). With its unique three-dimensional representation and analysis capabilities, this technology gives us a more real view of the world.

Useful Literature

Systems Systems Medicine – what it is and where we are

Dr Manuela Salvucci (RCSI)

Mathematical modelling has provided mechanistic insight into cell signalling and disease processes by identifying network-level control functions that originate from the interplay of multiple proteins. Validated systems models can accurately predict cell responses both kinetically and quantitatively. In translational studies, systems models also show potential for predicting for example tumour responsiveness to therapy. This talk will outline principles and concepts of systems biology research and illustrate the benefits of such approaches in the context of apoptosis signalling.

Useful Literature

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein

Mathematical modelling of the mitochondrial apoptosis pathway

Harnessing system models of cell death signalling for cytotoxic chemotherapy: towards personalised medicine approaches?

Polymorphisms Associated with Disease

Dr Ricardo Segurado (UCD)

Different strategies are required to identify rare and common genetic variants underlying both rare and common diseases. For common genetic variants, very large databases of known single nucleotide polymorphisms (SNPs) are usually used. These can be investigated using case-control studies or family-based studies, taking a candidate genes approach, or by whole genome association analysis, using hundreds of thousands of SNPs, and leveraging chromosomal proximity (linkage disequilibrium) and population history or genetic structure to impute many millions more. Recent developments include the use of High throughput sequencing of whole genomes which is revolutionising the clinical characterisation of rare disorders, and the development of polygenic risk scores from SNP genotypes as a catch-all proxy for risk of common conditions.

Useful Literature

Book: An Introduction to Genetic Epidemiology. Palmer, Burton & Davey Smith (Policy Press, 2011). The chapters were also published in the Lancet journal volume 266 (2005), in a series running from issue 9489: through to issue 9495.

Visscher et al (2017). 10 Years of GWAS Discovery: Biology, Function, and Translation. Am J Hum Genet 101(1):5-22.

Torkamani, Wineinger & Topol (2018) The personal and clinical utility of polygenic risk scores. Nature Reviews Genetics 19:581-590


Diagnostic challenges and proteomic solutions
Dr Holger Ebhardt (UCD)

Waiting rooms are full. Health care costs are rising. Will technological advances in analytical chemistry clear waiting rooms and lower long-term health care costs?

Dr Ebhardt will provide an introduction to proteomics using mass spectrometry, discuss discoveries of diagnostic protein biomarkers for patient surveillance in prostate cancer and diabetes, share his personal experience in not finding protein biomarkers for cancer cachexia, and close with an outlook on what the future of clinical diagnostics might look like.

Useful Literature

Ebhardt HA, Root A, Sander C, Aebersold R. Applications of targeted proteomics in systems biology and translational medicine. Proteomics. 2015, 15(18):3193-208. doi: 10.1002/pmic.201500004.

Rodriguez H, Pennington SR. Revolutionizing precision oncology through collaborative proteogenomics and data sharing. Cell. 2018, 173(3):535-539. doi: 10.1016/j.cell.2018.04.008.

Cellular Oxygen Consumption as an index of Metabolic Activity
Dr Richard Porter (TCD)


Cellular oxygen consumption gives a good measure of cellular metabolic activity. In primary cells, oxygen consumption is primarily due to oxidative phosphorylation by mitochondria. However, in some primary cells and cell lines a more comprehensive account of metabolism can be achieved by also measuring glycolytic flux. The Agilent Seahorse Flux Analyzer and the Oroboros Oxygraph Respirometer are two excellent and popularly used apparati to detect cellular oxygen consumption. In the former cells have to be adherent, whereas in the latter cells have to be in suspension. The lecture will shed light on the value of measuring cellular oxygen consumption.

Useful Literature

Porter RK, Brand MD.(1995) Cellular oxygen consumption depends on body mass, Am J Physiol. 1995 Jul;269(1 Pt 2):R226-8. PMID: 7631898

Geoghegan F, Chadderton N, Farrar GJ, Zisterer DM, Porter RK.(2017) Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells. Oncol Lett. 2017 Nov;14(5):6298-6306. doi: 10.3892/ol.2017.6929.

Martin DS, Leonard S, Devine R, Redondo C, Kinsella GK, Breen CJ, McEneaney V, Rooney MF, Munsey TS, Porter RK, Sivaprasadarao A, Stephens JC, Findlay JB.(2016) Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice. J Mol Endocrinol. 2016 Apr;56(3):261-71. doi: 10.1530/JME-15-0225.


Engineering replacement tissues and organs
Prof Daniel J Kelly (TCD)

This talk will review our attempts to use biomaterials and mesenchymal stem cells (MSCs) to engineer regenerative implants for use in orthopedic medicine. It will describe how 3D bioprinting can be used to engineer biological implants mimicking the shape of specific bones, and how these bioprinted tissues mature into functional bone organs upon implantation into the body. The talk will demonstrate how different musculoskeletal injuries can be regenerated using 3D bioprinted implants, including large bone defects and osteochondral defects. The talk will conclude by describing how we can integrate biomaterials and MSCs into 3D bioprinting systems to engineer scaled-up tissues that could potentially be used regenerate entire diseased joints.