Session 1

RNA Detection and quantitation
Dr Shane Duggan (Institute of Molecular Medicine, TCD)
The protein components of the cell are derived by numerous processes indirectly interpreted from a genetic element known as the “gene” which is coded in the cellular DNA. This element is interpreted by the cell in a process called “transcription” where the genetic code for a particular gene is converted into a molecular code known as messenger RNA (mRNA). This mRNA molecule can now be utilised in the creation of a new protein via the translation process. In this lecture the nature and analysis of Ribonucleic acid (RNA) in biological systems will be explored. The understanding of this has allowed the laboratory scientist to interrogate and explore gene expression as it may relate to diseases or cell signalling. Extraction and quantitation of good quality RNA will be discussed as they are the first step in any investigation of gene expression. Standard techniques in common use such as Northern blotting and cycle limited RT-PCR shall also be described as well as more modern techniques such as real time RT-PCR analysis. This lecture will allow the interpretation of published literature utilising these techniques and introduce the steps involved in performing RNA related techniques in your laboratory.

Differential gene expression: overview of relevant methods
Dr Karen Power (UCD Conway Institute)
This lecture will summarise the main approaches used to determine alterations in gene expression at the RNA level. Emphasis will be placed in this context on global approaches that attempt to map differences in the transcriptome, i.e. entire complement of transcripts in a cell. Methodologies that will be addressed include differential display, subtractive hybridization, SAGE, DNA microarray technologies and high-throughput sequencing. Relevant literature will be utilized to illustrate examples of investigators applying these technologies to understand biological phenomena, with a focus on cancer or other disease-related processes.

Review articles
A history of gene expression analysis:
Lennon, G. G. (2000). 'High-throughput gene expression analysis for drug discovery', Drug Discovery Today, 5, 59-66.
The use of profiling gene expression patterns:
Van’t Veer, L.J. and Bernards, R. (2008), 'Enabling personalized cancer medicine through analysis of gene-expression patterns', Nature, 453, 564-570.
Challenges faced in profiling tumour transcriptomes:
Coe, B.P., Chari, R., Lockwood, W.W. and Lam, W.L. (2008), 'Evolving strategies for global gene expression analysis of cancer', Journal of Cellular Physiology, 217: 590-7.
Experimental Design, Data preprocessing, Differential Expression Analysis, etc:
Grant, G.R., Manduchi, E. andStoeckert, C.J. (2007), 'Analysis and Management of Microarray Gene Expression Data' Current Protocols in Molecular Biology, Chapter 19:Unit 19.6.
Review of microarrays:
Schulze, A. and Downward, J. (2001), Navigating gene expression using microarrays – a technology review' Nature Cell Biology, 3, E190-E195.
Review on RNA-sequencing:
Wang, Z., Gerstein, M. and Snyder, M. (2009), RNA-Seq: a revolutionary tool for transcriptomics, Nature Reviews Genetics, 10, 57-63.

Polymorphism association with disease
Prof Denis Shields (UCD Conway Institute of Biomolecular & Biomedical Research)
Different strategies are required to identify rare and common genetic variants underlying both rare and common diseases. For common genetic variants, there is now a very rich dataset of identified common single nucleotide polymorphisms (SNPs). These can be investigated in disease groups (compared to controls) in candidate genes, or by whole genome association analysis, using chips with one million different SNPs on them. Analysis of these variants requires careful attention to the patterns of association of SNPs that are chromosomally adjacent (in linkage disequilibrium, meaning that the variant at one site is associated with a particular variant at another site, arising from the population history of the origin of the mutation, its spread through the population, and the gradual breaking down of the association of adjacent variants by recombination events). Linkage analysis (tracking in families the disease co-inheritance with widely spaced gene markers) is the traditional approach of choice for rare mutations that have strong phenotypic effects. High throughput sequencing of candidate regions (and in future whole genomes) are accelerating the rate of data accumulation.

Model organisms
Dr Yolanda Alvarez (UCD School of Biomedical and Biomolecular Sciences, UCD Conway Institute)
The goal of this lecture is to discuss animal models that are routinely applied to biomedical research. The advantages of using Drosophila (fly), Xenopus (frog), Danio (zebrafish), Gallus (chicken) and Mus Musculus (mouse) as model organisms will be described. The life-cycle, generation time, embryo development and amenability of these organisms to genetic manipulation will be discussed. An emphasis will be placed on describing mutagenesis screens. This technique, in which the genes in the genome are randomly inactivated, has been extensively applied to the fly/fish models and has accelerated our understanding of gene function (functional genomics). 

Session 2

DNA cloning strategies
Dr Ross McManus (Institute of Molecular Medicine, TCD)
Even in the post genome era, DNA cloning is essential to the manipulation and stable propagation of genetic material. This talk will cover the basic aspects of DNA cloning, ranging from the anatomy of cloning vectors to the choice of vectors based on the cloning strategy employed. The strategy employed will depend on the overall objectives of the project and the nature of the starting information or material available. Thus different choices and approaches would be employed for a sequencing project compared with a genome mapping project or production of RNA or protein. I will discuss a number of basic and specialised cloning strategies to illustrate some of the options and possibilities available.

Transgenics and knockouts
Dr Rosemary Kane (UCD)
This lecture will provide a broad overview of the strategies used to generate both transgenic and knockout mice, starting from the generation of the DNA constructs using cDNAs or genomic DNA, and proceeding through embryonic stem cell biology, to aggregation and chimeric mouse generation. Details on genotyping of transgenic animals, as well as phenotype characterization will be discussed. Specific examples will be cited. 

Determining protein:protein interactions in biology
Prof Niamh Moran (RCSI Research Institute)
Whereas the Human genome Project has successfully identified all human genes and their corresponding proteins, the next challenge in biology is to understand how these proteins interact with each other in a dynamic cell system. My lab is interested in the molecular mechanisms of integrin activation in the human platelet in order to better understand thrombotic disease. Using the platelet as a model system therefore, we explored various mechanisms of elucidating relevant protein interactions with integrin cytoplasmic tails. Current conventional methods are discussed in this presentation including co-immunoprecipitation, FRET, yeast-two hybrid system, TAP-Tags and protein chip technology. In addition, the emerging protein interaction websites provide a useful tool in this process. Verification of newly identified interactions is the next bottle-neck in the process and needs to be tailor made for each specific protein. This involves a combination of literature-searching, bio-informatics and basic cell-biology techniques. The conclusion of this presentation will highlight the importance of choosing an appropriate system for exploring protein:protein interactions; identifying relevant association and eliminating non-specific interactions, and finally emphasising the importance of relevant verification in this process. We have termed this process Protein ENONOMICS from the Greek term for interaction and claim that it is the next emerging ‘omic’ after proteomics!
 

Session 3

Protein expression and purification
Dr Henry Windle (Institute of Molecular Medicine, TCD)
This lecture 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. As protein purification methodologies are generally well described and accessible, only a brief overview of these will be given but with emphasis on common problems that can arise, particularly for those about to attempt purification for the first time.
The following books from The Practical Approach series by IRL Press are an invaluable aid with detailed and reliable protocols:
 Protein Purification Applications; Protein Purification Methods (2001, Editor Simon Roe).
The always excellent Methods in Enzymology has just published a comprehensive overview of protein expression and purification (Methods in Enzymology 463, 1-851 (2009): RR. Burgess and M P. Deutscher, Eds).
 

Introduction to Mass Spectrometry
Dr Giuliano Elia (UCD Conway Institute of Biomolecular & Biomedical Research)
Mass spectrometry is a powerful technique for the precise and accurate determination of the molecular mass of a vast range of chemical compounds. The introduction, about two decades ago, of soft ionization techniques like MALDI (Matrix-Assisted Laser Desorption-Ionization) and ESI (ElectroSpray Ionization) allowed the widespread application of mass spectrometry to biological macromolecules, including proteins and peptides. Mass spectrometry is truly at the origin of the enormous success of proteomic science.
This lecture will present basic concepts in mass spectrometry, introduce the most common types of mass spectrometers used in biological research and discuss some simple mass spectrometry-based proteomic approaches.

Expression proteomics: biomedical applications
Prof. Stephen Pennington (UCD Conway Institute of Biomolecular & Biomedical Research)
Following the sequencing of the genomes of a large number of organisms including the landmark publications of the human genome it has become increasingly apparent that the study of their encoded proteins - on a genome-wide scale - is required. This is the field of proteomics. When, in 1994, Marc Wilkins coined the term ‘proteome’, defined as the protein complement of a genome, the methods for investigating protein expression on such a scale were in their infancy and today (a decade and a half later) they are still in rapid evolution.

The technique of two-dimensional gel electrophoresis (2-DE) remains a core for many applied proteomic projects due to its ability to separate simultaneously thousands of proteins and to indicate post-translational modifications that result in alterations in protein pI and/or Mr. Moreover, recent developments for 2-DE including the use of fluorescent dyes that facilitate the multiplex analysis of samples make it possible to achieve greater proteomic coverage combined with more accurate differential expression analysis.

Liquid chromatography (LC) based methods have also emerged as powerful approaches for protein expression analysis. Multi-dimensional LC approaches potentially have the advantage of higher coverage and sensitivity but are only recently being applied to the routine separation and quantification of very complex mixtures of proteins. In this presentation several biomedical applications will be described to show the utility of gel and LC based proteomics approaches including the use of isotope coded affinity tags. Together, these should illustrate the diversity of approaches available for the measurement of protein expression.

Review article
Cravatt BF, Simon GM, Yates JR 3rd. The biological impact of mass-spectrometry-based proteomics. Nature. 2007 Dec 13;450(7172):991-1000. Review.
PMID: 18075578 [PubMed - indexed for MEDLINE]

Immunodetection methods on cell and tissue extracts
Dr Leonie Young (RCSI)
The use of antibodies to detect and characterise proteins has been well established. With the development of high through-put techniques such as tissue microarrays (TMA), a real challenge now exists to determine the cellular location, level of expression and the function of these identified proteins. In this lecture, principles fundamental to immunodetection will be outlined. Common pitfalls and measures to avoid these will be discussed. 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.

Cell imaging and sorting – flow cytometry
Prof. William Watson (UCD Conway Institute of Biomolecular & Biomedical Research)
Flow cytometry is a method for quantitating components or structural features of cells, primarily by optical means. Although it makes measurements on one cell at a time, it can process thousands of cells in a few seconds. Since cell types can be distinguished by quantitating structural features, flow cytometry can be used to count prokaryotic or eukaryotic cells of different types in complex mixtures.

Flow cytometry overview (PDF) 

Session 4

Imaging using Fluorescent/Confocal Microscopy
Dr Orla Hanrahan (TCD)
Nowadays, 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.

Suggested reading material:
Microscopy Primer Website http://micro.magnet.fsu.edu/primer/index.html (very good site with tutorials)
Handbook of Biological Confocal Microscopy, edited by James B. Pawley
Protein Localisation by Fluorescence Microscopy, edited by V.J. Allan, Practical Approach Series

High Content Screening and Analysis Platform technologies
Dr Anthony Davies (Institute of Molecular Medicine, TCD)
High Content Screening and Analysis (HCS(A)) is becoming synonymous with the reporting of biochemical events occurring within individual cells. This new technology now permits visualisation and quantitation of a wide range of target-specific responses on a per cell basis. HCA technologies are now capable of providing a highly detailed picture of a cells physiological state. This technology allows for the first time a quantitative assessment of gross morphology, sub-cellular target localisation, cell viability, apoptosis and cytoskeletal rearrangement of single cells in populations.
Here at IMM Trinity we have already successfully utilised HCA technologies in cell-based cancer, inflammation and infection cardiovascular and immunity studies.
In the Techniques and Strategies in Molecular Medicine course, we will cover the fundamental aspects of HCA technology deployment and utilisation. This will form a solid foundation, on which to build further knowledge and experience in this new and exciting scientific discipline.

Stem Cells - Biology and Applications
Dr Linda Howard (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 human cells to study development and to look at human-specific toxicity testing if drugs.
This lecture will describe stem cells sources, characteristics and properties. Finally it will cover ongoing clinical trials designed to evaluate the safety and efficacy of stem cell therapies.

Other sources of reading
Online book on stem cells (free)
http://stemcells.nih.gov/info/scireport/2001report.htm

Online book on regenerative medicine (free)
http://stemcells.nih.gov/info/scireport/2006report.htm