Improving in situ hybridisation discussion forum - 29 November 2012

Improving in situ hybridisation discussion forum

The Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage, Herts, UK: 29 November 2012

This technical workshop is dedicated to the discussion of in situ hybridisation (ISH), its capabilities, difficulties and troubleshooting. Invited speakers will give an overview of their area of expertise in the morning session, with the afternoon devoted to informal round table discussions.

This event will be an ideal place to network, establishing lines of communication for the future if you ever need help with setting up the technique and giving you short cuts to improving your results.

Why re-invent the wheel when you can learn how to do it here?

Having trouble setting up the technique? Specific questions will be answered, which kits to use, which concentrations to use.

Why you should come: This is a specialist meeting, with no other forum comparable internationally, as it is rare to have so many experts in the field, together in one room, giving you unrivalled access, all in one place.

Who should attend: PhD students, postdocs, technicians and any scientists trying to set up ISH or already running it. Even experienced people who are interested in keeping up with the technique should be at this unique event.

Meeting Chair: Dr Julia Jones, Cancer Research UK, Cambridge

This event has CPD accreditation

9:00 – 9:30 Registration

9:30 – 9:35 Introduction by Meeting Coordinator: Dr Astrid Englezou , Euroscicon, London, UK

9:35 – 9:40 Introduction by the Chair: Dr Julia Jones, Cancer Research UK, Cambridge

9:40– 10:00 Probing for the invisible –Detecting micro-RNAs in tissues
Dr Julia Jones, Histopathology/ISH Core Facility, Cancer Research UK,Cambridge Research Institute,UK
Micro-RNA (miRNAs) are small non-coding RNAs that regulate the expression of other genes. miRNAs have influence over diverse biological functions and have been found to contribute to disease, including cancer. The study of miRNAs is complicated by a number of factors including mature miRNA length and high degrees of homology between miR families. We have developed a protocol for visualising miRNA in FFPE tissue sections using in-situ hybridisation and this methodology will be described.

10:00 – 10:20 RNA in situ hybridization
Dr James Howard Pringle, Reader in Molecular Pathology, Department of Cancer Studies and Molecular Medicine, University of Leicester
Although the technique of RNA in situ hybridization (ISH) was developed over 40 years ago it has not the same prominence in the literature as immunohistochemistry. This could be due to the fact that this technique is difficult to optimize as cells and tissues samples must be suitably handled, stored and fixed to preserve RNA to achieve optimal results. This presentation will describe the factors influencing the quality of RNA ISH using non-isotopic labelled probes. Examples will be used to demonstrate the important parameters that influence this technique as well as the choice of appropriate controls. The talk will initially cover the methods for handling cells and tissue samples. The ISH technique will be examined including; target gene validation, probe design, labelling, hybridization conditions, detection methods and signal evaluation. Is this technique sufficiently robust with the sensitivity and specificity needed to detect and localise gene expression in human and animal tissues?

10:20 – 10:40 FISH on DNA targets from chromosomes to DNA fibres

Paul Edwards, Hutchison-MRC Research Centre, Cambridge, UK

FISH can be used on a range of DNA targets. For classical chromosome spreads, multicolour FISH with whole-chromosome probes can be used on whole karyotypes, while band-specific probes and BAC or smaller probes show rearrangements within chromosomes. FISH can be used on interphase nuclei on slides or in paraffin sections, or stretched fibres of DNA, giving much higher resolution than classical cytogenetics and measuring distances down to perhaps 10kb. In paraffin sections, including tissue microarrays, chromosome rearrangements can be detected in tumours. Fluorescence can also be replaced by histochemistry to give Chromogenic In-Situ Hybridization or CISH.

10:40 – 11:00 Mid-morning Break

Please try to visit all the exhibition stands during your day at this event. Not only do our sponsors enable Euroscicon to keep the registration fees competitive, but they are also here specifically to talk to you

11:00 – 11:20 Expert Talk to be confirmed

Dako

11:20 – 11:40 Qdots for ISH

Richard Byers, University of Manchester, UK

Though protein immunohistochemistry is the easiest method for visualisation of gene expression, antibodies are not available for all proteins, in which cases mRNA expression analysis, by ISH, is required. QDs have been used extensively for in situ mRNA detection by ISH. This talk will cover the use of QDs for ISH, starting with early applications and covering duplex and triplex examples from the presenter’s work and from the literature. Some groups have sued QDs for combined protein and mRNA detection and examples of these studies will be presented. Early studies were performed in fresh or frozen material but the presenter’s group has extensive experience in QD-ISH in formalin fixed samples, which are key for translational studies. Different conjugation chemistries will be outlined and examples of application to chromosomal analysis presented.

11:40 – 12:00 Isotopic ISH: why is it still useful?

Professor Richard Poulsom, Blizard Institute, Queen Mary, University of London, UK

Isotopic ISH to localise expression of specific mRNAs has been used for several decades to great effect. Non-isotopic methods have gained popularity, but do they always give the same results, and if not why not? I will explore the advantages and disadvantages of various methods to help you choose which best suits your experimental needs.

12:00 – 12:30 Working Lunch

Please collect your lunch and take it to your discussion table (Session 1)

This is also a good time to fill out your feedback forms

12:30 – 14:10 Discussion Group Sessions 1 - 3

• Round table discussion groups (20 minutes each) will be held throughout the afternoon
• Delegates will rotate so that they may participate in all the discussion tables
• All delegates will also be allocated a session for visiting the exhibition stands
• Where appropriate delegates will be able to bring their samples to the discussions
• See end of agenda for description of discussion tables

14:10 – 14:30 RNAscope - Universal Assay for RNA in situ Hybridisation providing Single Copy Sensitivity

Dr Kai Wilkens, Advanced Cell Diagnostics, Inc, USA

ACD is a start-up molecular pathology company in the SF Bay Area who has developed a novel assay platform called RNAscope for RNA in situ hybridization (ISH). It is a proprietary, breakthrough technology for RNA ISH that is useful for gene expression analysis in fixed and fresh-frozen tissue. RNAscope offers an ultra-sensitive, clinically proven and universal assay for any expressed gene in situ. It detects single RNA molecules that appear as “spots” with RNAscope. The talk will show the details of the technology, validation data and application examples.#

14:30 – 15:00 A Novel Multiplex RNA in situ Hybridization Platform that Enables Single Copy Detection in Cells and Tissues

with Spatial Resolution

Dr Paul R Turner, Affymetrix UK Ltd.

RNA In Situ Hybridization (RNA ISH) is an important technique for biomarker guided drug development, medical oncology and cytologic and anatomic pathology. Newly discovered RNA transcripts are being validated to generate new classes of prognostic and predictive diagnostic tests. In addition, RNA ISH assays serve an important role in pathology and translational research by complementing immunohistochemistry (IHC), for example in cases where antibodies perform poorly or are unavailable as is the case for non-coding RNAs (ncRNAs), and the detection of cells expressing secreted proteins. RNA ISH can specifically localize RNA targets within cells, including Circulating Tumor Cells (CTCs) and in fresh-frozen and Formalin Fixed Paraffin Embedded (FFPE) tissues and tissue microarrays (TMA’s)

However, over the years RNA ISH has seen little significant progress until the recent commercialization of the QuantiGene ViewRNA ISH platform. This technology is based on branched DNA reporter amplification and uses highly specific probe sets that enable multiplexed single copy detection of mRNAs and ncRNAs with spatial resolution within cells and tissues.

The QuantiGene ViewRNA platform is being developed into a suite of Research Use Only (RUO) validated oncology FFPE tissue biomarker assays. These will combine standardized RNA CISH & FISH kits with Leica Bond RX sample preparation and staining automation, and will use Flagship Biosciences digital image analysis, producing a seamless start-to-finish RNA ISH solution. QuantiGene ViewRNA RUO assays will be translated into clinical diagnostic tests.

This presentation will focus on QuantiGene RNAView ISH Assays in a wide variety of sample types, including cells and tissues, and will include both singleplex and multiplex results . Finally, the importance of RNA expression heterogeneity in disease and model systems will be discussed.

15:00 – 16:00 Discussion Group Sessions 4 - 6

16:00 – 16:40 Question and Answer Session

This session will include summing up of the discussion tables and questions submitted both prior to the meeting and throughout the day

16:40 – 16:45 Chairman’s Summing Up and Feedback Prize Draw

Discussion tables

Table A: Julia Jones

Table B: Howard Pringle

Table C: Paul Edwards

Table D: Richard Byers

Table E: Dako

Table F: Richard Poulsom

About the chair
Julia Jones graduated with a BSc (Hons) in Biomedical Science from the University of Southampton before gaining a PhD in Neuroscience from Cambridge University. She is now with Cancer Research UK as a Senior Scientific Officer (ISH) where she has run the ISH service in the Histopathology/ISH facility at the Cambridge Research Institute for 5 years.

About the speakers
Howard Pringle graduated from Bristol (1979) in Applied Biological Sciences and completed a PhD in Biotechnology at the University of Warwick (1983). He studied DNA replication as a post-doctoral scientist in the Department of Molecular Biology, University of Edinburgh. Then moved to University of Leicester in 1985 as Non-clinical Lecturer in the Department of Pathology. He is currently a Reader in Molecular Pathology in the Department of Cancer Studies and Molecular Medicine at Leicester. His research includes molecular studies of melanoma, breast and colorectal cancer. He has developed and applied non-isotopic in situ hybridisation methods on human and animal tissues since moving to Leicester in 1985

Richard Pousom, together with a team of expert colleagues, operated a core facility for localisation of mRNAs for many years; looking at the expression of hundreds of mRNAs in around 89,000 sections of routinely fixed and processed human and experimental tissues, mostly using 35S and 3H riboprobes. Recently he has been exploring the newer signal amplifying non-isotopic ISH methods with the intention to compare their results to isotopic ISH and to combine them with immunohistochemistry.

Kai Wilkens has spent some 20 years in the pharmaceutical and Life Science markets. Before joining ACD he managed sales teams for MSD and Affymetrix and supported customers in the biomarker field. Prior to this he headed the global marketing of MWB Biotech AG, a startup company in the areas of oligonucleotide synthesis and sequencing services, mircoarrays and liquid handling instrumentation. Before that Dr. Wilkens worked as a CRA in clinical research for Ardeypharm. Dr. Wilkens received his Ph.D. in Microbiology from Ruhr-University Bochum and a MBA from the FOM University for Economy and Management.

Paul Edwards: Faculty position in Cambridge for nearly 30 years. Since early 90s research has focussed on genomics and molecular cytogenetics of breast cancer, particularly searching for chromosome translocations and fusion genes. Extensive experience of FISH on DNA targets, particularly with BAC probes, chromosome painting and SKY (24-colour FISH karyotyping). Now moving into massively parallel sequencing.
Richard Byers, After general medical training, including an intercalated BSc in Anatomy and an MSc in Cell and Structural Biology from The University of Manchester, I trained as a histopathologist and completed a PhD in molecular biology, specifically in bone biology, in 1999. Since then I have developed a diagnostic and research interest in haematopathology; my specialist diagnostic clinical interest is in leukaemia and lymphoma. I am a senior lecturer in Pathology in the School of Cancer and Enabling Sciences at The University of Manchester and Consultant Haematopathologist at Manchester Royal Infirmary. I am also Head of Service for Greater Manchester and Cheshire Haematological Malignancy Diagnostics, which provides an integrated diagnostic service, including molecular diagnostics, in leukaemia and lymphoma for the Greater Manchester and Cheshire Cancer Network. In addition, I am a molecular pathologist for the Clinical Experimental Pharmacology Group within the Paterson Institute for Cancer Research.

I was visiting professor at the Broad Institute of Harvard and the Massachusetts Institute of Technology (MIT) between 2003 and 2004 and continue to collaborate with the group. I was Chair of the Molecular Pathology Group of the Pathological Society of Great Britain and Ireland and have given several invited lectures on Qdot in-situ hybridisation and immunohistochemistry. I have authored or co-authored around 35 publications including manuscripts, book chapters and review articles and am a reviewer for the Journal of Pathology, Histopathology, British Journal of Haematology and Journal of Clinical Pathology.

Keywords: RNA, in situ hybridization, nucleic acid probes, non-isotopic labels, ISH, FISH, miRNA, 35S riboprobes, tyramide enhancement, miRNA detection, CISH, FISH, DNA, breast cancer, IHC validation, RNAscope, digital pathology, gene expression