GeneExpression Systems of USA and University of Cambridge of UK
Jointly Presents
Second International
MicroRNAs Europe 2007 Meeting
on
MicroRNAs: Biology to Development and Disease

November 1-2, 2007
Venue: Peterhouse, University of Cambridge, Cambridge, UK

A Unique Theme Conference & Exhibition in the MicroRNomics!

Meeting Place:
The Venue Peterhouse (middle) is the oldest College of the University of Cambridge, founded in 1284 and an institution dedicated to education and research. Throughout its history, Petreans have been at the heart of the political, social and religious controversies that have shaped today’s society. Some of the influential Petreans include: Henry Cavendish, Lord Kelvin, Sir Frank Whittle and Christopher Cockerill, and Nobel Laureates – Sir John Kendrew, Sir Aaron Klug, Archer Martin and Max Perutz , who gave a twentieth century lead in the field of Molecular Biology. Since many centuries it remained as a hub for innovation and successive generations of the brightest young people around the world.

Why do you wait to join for an intellectual gathering in the ‘microRNomics’ arena at the prestigious college Peterhouse in the Premier University campus!

Target Audience: 200
Total Speaker Presentations: 20
Total Poster Presentations: 20
Total Exhibit Booths: 20

AGENDA/SPEAKERS

Click Here For Agenda

Thursday, November 1, 2007
7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 A.M: Continental breakfast
8:00 - 9:30 A.M: Technology Session I (consists of 3 lectures)
Friday, November 2, 2007
7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 A.M: Continental breakfast

8:00 - 9:30 A.M: Technology Session II (consists of 3 lectures)
Scientific Sessions Start at 9:30 A.M and Ends at 6.00 P.M on all two days.
The actual agenda will be updated. Please visit again.

Organizers:

Krishnarao Appasani, PhD., MBA (Chair)
Founder & CEO
GeneExpression Systems, Inc. Waltham, MA USA

Sakari Kauppinen, Ph.D.
Associate Director
Santaris Pharma, Copenhagen, Denmark

Anton J. Enright, PhD.
Group Leader of Computational and Functional Genomics
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom

Eric A. Miska, PhD.
Assistant Professor at the Wellcome Trust/Cancer Research UK/Gurdon Institute
University of Cambridge, Cambridge, United Kingdom

Peter Roberts, PhD.
Manager
Febit GmbH, Heidelberg, Germany


Inaugural Speaker Speaker on November 1

Sir Peter Mansfield, FRS Nobel Laureate 2003 Emeritus Professor of Physics, University of Norttingham, Nottingham, UK Title: Early Developments in Magnetic Resonance Imaging (MRI)

Keynote Speaker on November 1 (MicroRNAs Innovator Award)

David Baulcombe, PhD., FRS Royal Society Professor University of Cambridge, Cambridge, UK Title: TBA

Keith McCullagh, PhD BVSc MRCVS President & CEO Santaris Pharma, Horshol, Denmark Title: TBA

Other Speakers:

Minoo Rassoulzadegan, Ph.D.
Director-INSERM, Faculty of Sciences
University of Nice, Nice, France
Title: RNA-mediated induction of hereditary epigenetic modification

Ramin Shiekhattar, PhD.
ICREA Professor
CRG -Centre de Regulació Genòmica, Barcelona, Spain
Title: TBA

Thomas Thum, MD.
Head, Research Group Cardiac Wounding/Healing,
Julius-Maximilians-University Würzburg, Germany
Title: MicroRNAs in human end-stage heart failure

Frank Opdam
Head of R & D
Kreatech Biotechnology B.V. Amsterdam, The Netherlands
Title: ULS labeling of miRNA is strongly down-scalable and compatible with many platforms

Annick Harel-Bellan, PhD.
Director of Laboratoire "Epigenetique et Cancer"
Institut Andre Lwoff, Villejuif France
Title: Micro-RNAs and muscle differentiation

Tamas Dalmay, PhD.
Lecturer in Cell and Molecular Biology
University of East Anglia, Norwich, UK
Title: The role of microRNAs in cartilage development

Pål Sætrom, PhD.
Interagon, Trondheim, Norway &
Department of Cancer Research and Molecular Medicine
Norwegian University of Science and Technology, Norway
Title: Distance Constraints Between MicroRNA Target Sites Dictate Efficacy and Cooperativity

Greg Arndt, PhD.
Research Director, Antiviral Target Discovery Group
Johnson & Johnson Research, Eveleigh, Sydney, Australia
Title: MicroRNA Expression Profiling and Functional Analysis of Specific MicroRNAs in Colorectal Cancer

Joost P.G. Sluijter, PhD.
Post doctoral Fellow, Department of Cardiology
University Medical Center, Utrecht, The Netherlands
Title: microRNA regulation in cardiac regeneration

Peter Roberts, PhD.
MicroRNAi Product Manager
Febit Biotech GmbH, Heidelberg, Germany
Title: Microfluidics and microRNA: a customisable platform for profiling small RNAs

Martin Turner PhD.
Head of Laboratory of Lymphocyte Signalling & Development
The Babraham Institute
Babraham Research Campus
Babraham, Cambridge CB22 3AT, UK
Title: Regulation of immunity by bic/microRNA155

Cesare Peschle, M.D.
Professor and Chairman
Dept. of Hematology, Oncology and Molecular Medicine
Istituto Superiore di Sanità, Roma, Italy
Title: MicroRNA control of heart hypertrophy and normal hematopoiesis

Carsten Alsbo, Ph.D.
Product Manager
Exiqon A/S, Denmark
Title: Discovery of microRNA cancer biomarkers - using miRCURY™ LNA Array

Applied Biosystems sponsored speaker
Finbarr E. Cotter, MB, BS, FRCP( UK ), FRCPath, PhD
Professor of Experimental Hematology
Queen Mary's School of Medicine and Dentistry,
London, United Kingdom
Title: To Be Announced

Raghu Vemuganti, PhD
Assistant Professor of Neurological Surgery
University of Wisconsin, Madison WI, USA
Title: MicroRNA profiles in Cerebral Ischemia: Key players in post-ischemic gene regulation

James K. Roberts, PhD.
Gene Suppression Team Lead
Monsanto, Chesterfield, MO, USA
Title: Control of coleopteran insect pests through RNA interference

Aristotelis Tsirigos, PhD.
Computational Biology Center
IBM Thomas J Watson Research Center
Yorktown Heights, NY, USA
Title: Non-conserved intronic motifs are related to piRNAs and linked to a conserved set of functions in human and mouse

Pramod Upadhyaya, Ph.D.
Sponsored By Sigma-Life Science, UK-USA
Assistant Professor
Department of Pharmacology and Cancer Center, University of Minnesota
Minneapolis, MN, USA
Title: MicroRNA Expression in a Carcinogen-Induced Cancer Model

Larry A. McReynolds, PhD.
Director of RNA Biology
New England Biolabs, Ipswich, MA, USA
Title: Affinity purification of small RNAs with plant viral protein p19

Taro Fukao, PhD.
Medical Fellow
Tokyo Medical and Dental University, Tokyo, Japan
Title: MicroRNA-223 expression by gene profiling

Petula D’Andrade, PhD.
Agilent Product R&D Labs Specialist
Agilent Technologies
Title: A microarray platform for miRNA profiling that is both sensitive and robust

Sylvia Schuetz, PhD.
Postdoctoral Fellow in the Lab of Professor Peter Sarnow
Stanford University Medical School, Stanford, CA, USA
Title: Hepatitis C virus replication depends on liver-specific microRNA miR-122

Anita G. Seto, Ph.D.
Research Scientist
ThermoFisher Scientific, Dharmacon Products, Lafayette, CO, USA
Title: TBA

Karen P. Steel, PhD.
Investigator
Wellcome Trust Sanger Institute
Hinxton, Cambridgeshire, UK
Title: MicroRNA expression in hearing impairment

Eugene Berezikov, PhD.
Group Leader, Hubrecht Institute, Utrecht, The Netherlands, &
Co-founder
InteRNA Genomics, Bilthoven, The Netherlands.
Title: Discovery and expression profiling of small RNAs by deep sequencing

Simon Mauch, PhD.
Global Marketing Team Manager
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Title: TBA
Each speaker will have 20 min for presentation and 5 min for discussion.

Panel Discussion on November 2, with experts from:
- Venture Capital Firm
- Technology Transfer Office
- Professional Science/Business Journalists
- Patent Attorney from a Law Firm
and selected speakers from the conference.

Key Sessions:
The most-up-to-date developments will be addressed:
MicroRNAs Discovery and Biogenesis
MicroRNAs in Development
Bioinformatics of miRNAs
MicroRNAs in Virology & Diagnostics
MicroRNAs in Stem Cell Biology
MicroRNAs in Disease Biology

Exhibitors are welcome to reserve their booth space early!

Please contact if you are interested in speaking in the scientific or Technology workshops of this meeting.

GeneExpression Systems, Inc.
P.O. Box 540170
Waltham, MA 02454 USA
Tel: (781) 891-8181
Fax: (781) 891-8234
E-mail: Genexpsys@expressgenes.com
www.expressgenes.com

Poster Abstract Submission by October 15, 2007

ALL ABSTRACTS

Early Developments in Magnetic Resonance Imaging (MRI)
Peter Mansfield, PhD., FRS. Emeritus Professor of Physics, University of Norttingham, Nottingham, UK

Following an initial description of the early days in MRI the talk will concentrate on patient safety aspects by discussing some novel developments in the control of acoustic noise and also the control of electric fields.

MicroRNAs in the Human Heart: A Clue to Fetal Gene Reprogramming in Heart Failure
Thomas Thum 1,2*, M.D., Junior Group Leader at the Julius-Maximilians University
Department of Cardiology, University of Würzburg, Würzburg, Germany

Thomas Thum 1,2*, M.D., Paolo Galuppo1, Ph.D., Susanne Kneitz3, Ph.D., Jan Fiedler1,2, Christian Wolf1, Linda W. van Laake4, M.D., Pieter A. Doevendans4, M.D., Ph.D., Christine L. Mummery4, Ph.D., Carina Gross5, Stefan Engelhardt5, M.D., Ph.D., Georg Ertl1, M.D., and Johann Bauersachs1, M.D.
1University of Würzburg, University Hospital, Department of Internal Medicine I, Cardiology, Würzburg, Germany
2University of Würzburg, Interdisciplinary Center for Clinical Research (IZKF), Junior Research Group Cardiac Wounding and Healing, Würzburg, Germany
3University of Würzburg, Interdisciplinary Center for Clinical Research (IZKF), Microarray Core Facility, Würzburg, Germany
4Hubrecht Laboratory and the Heart Lung Institute, University of Utrecht Medical Center, Utrecht, Netherlands
5University of Würzburg, Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, Würzburg, Germany

Background: Chronic heart failure is characterized by left ventricular remodeling and reactivation of a fetal gene program; the underlying mechanisms are only partly understood. Here we provide evidence that cardiac microRNAs, recently discovered key regulators of gene expression, critically determine the transcriptional changes observed in heart failure.
Methods and Results: Cardiac transcriptome analyses revealed striking similarities between fetal and failing human heart tissue. Using microRNA-arrays we discovered profound alterations of microRNA expression in failing hearts. These changes closely mimicked the microRNA expression pattern observed in fetal cardiac tissue. Bioinformatic analysis demonstrated a striking concordance between deregulated messenger RNA expression in heart failure and the presence of microRNA binding sites in the respective 3´untranslated regions. Messenger RNAs upregulated in the failing heart contained preferentially binding sites for downregulated microRNAs and vice versa. Mechanistically, transfection of cardiomyocytes with a set of fetal microRNAs induced cellular hypertrophy and disarray as well as changes in gene expression comparable to the failing heart. A variety of further in vivo and in vitro approaches are on the way to improve our understanding in the regulation of gene networks by miRNAs in heart failure. Conclusions: Our data support a novel mode of regulation for the transcriptional changes in cardiac failure. Reactivation of a fetal microRNA program substantially contributes to alterations of gene expression in the failing human heart.

Distance Constraints Between MicroRNA Target Sites Dictate Efficacy and Cooperativity
Pål Sætrom, PhD., Scientist, Interagon, Trondheim, Norway

MicroRNAs have the potential to regulate the expression of thousands of genes, but the mechanisms that determine whether a gene is targeted or not are poorly understood. We studied the genomic distribution of distances between pairs of identical microRNA seeds and found a propensity for moderate distances greater than about 13 nucleotides between seed starts. Experimental data show that optimal downregulation is obtained when two seed sites are separated by between 13 and 35 nucleotides. By analyzing the distance between seed sites of endogenous microRNAs and transfected siRNAs, we also find that cooperative targeting of sites with a separation in the optimal range can explain some of the siRNA off-target effects that have been reported in the literature.

MicroRNA control of heart hypertrophy and normal hematopoiesis
Cesare Peschle, M.D., Professor and Chairman, Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Growing evidence indicates that microRNAs (miRNA) are involved in basic normal and abnormal cell functions. Our heart studies show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease. Our studies on hematopoiesis indicate that: a) miRNA-221/222 downmodulated in early hematopoiesis and in erythropoiesis, control the level of c-kit (receptor of the stem cell factor), thus regulating hematopoietic stem cell activity and early erythroid proliferation. b) Monocytopoiesis is controlled by a circuitry involving sequentially downmodulation of miRNA 17-5p-20a-106a and upmodulation of the AML1 target, leading to upregulation of M-CSF receptor, whereby miRNA 17-5p-20a-106 function as a master gene complex interlinked with AML1 in a mutual negative feedback loop.

microRNA regulation in cardiac regeneration
Joost P.G. Sluijter, PhD., Department of Cardiology, Experimental Cardiology Laboratory, Utrecht, The Netherlands

Joost P.G Sluijter, Pieter A.F. Doevendans, Marie-José Goumans

Improving the intrinsic regenerative capacity of the heart and/or restore damaged tissue will minimize cardiac deterioration. Recently, the contribution of microRNA (miRNA) regulation during heart development and cardiac pathology was identified. We studied the role of miRNA regulation after cardiac injury in rodents and in proliferation/differentiation of human cardiomyocyte progenitor cells (CMPC). The potential role of miRNAs during different phases of cardiac regeneration was studied. We will discuss the role/function of miRNAs that are studied in cardiac tissue before and new unexplored miRNA for their potential function in cardiac regeneration, pointing to a strong regulatory role of miRNAs.

Microfluidics and microRNA: a customisable platform for profiling small RNAs
Peter Roberts, M.Sc., Ph.D., microRNAs Product Manager, Key Account Manager Nordics and BeNeLux, Febit biotech, GmbH, Heidelberg, Germany

febit’s Geniom platform provides an ideal tool for scientists to design their own microarrays for miRNA analysis. Based on microfluidics, it allows customisable in situ synthesis of oligo arrays, both from publicly available and proprietary sequence information, directly by the user. Therefore, the Geniom technology not only allows high-throughput miRNA quantification, but also the verification of newly predicted miRNAs. Here we present data showing the high reproducibility and accuracy of miRNA expression profiling data obtained with the Geniom platform and also suggest an optimized workflow which provides scientists with a flexible, accurate and easy-to-use tool for miRNA analysis.

Micro-RNAs and muscle differentiation
Annick Harel-Bellan, PhD., Director, Laboratoire "Epigenetique et Cancer", FRE 2944 CNRS, Institut Andre Lwoff, 94800 Villejuif France

Irina Naguibneva and Annick Harel-Bellan
UPR 9079 CNRS, Institute Andre Lwoff, 7 rue Guy Moquet, 94800 Villejuif, France.

MicroRNAs are essential for development, and for the control of cell proliferation/differentiation in various organisms. We examined the micro-RNA expression during terminal differentiation of a myoblastic cell line (C2C12), or embryonic precursors (ES cells) forming muscle-oriented embryonic bodies in culture. MiR-181 has been found among the most strongly up-regulated miRNAs when differentiation is induced in these two differentiation models. MiR-181 was barely detectable in adult muscle, but, surprisingly, strongly up-regulated upon regeneration of muscle fibers. Our results suggest that miR-181 downregulates the homeobox protein Hox-A11, a repressor of the differentiation process. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype – even when they are not expressed in the corresponding fully differentiated tissue.

Regulation of immunity by bic/microRNA155
Martin Turner PhD., Head of Laboratory of Lymphocyte Signaling & Development
The Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB22 3AT, UK

Martin Turner, Susan Kohlhass, Antony Rodriguez, Allan Bradley, Cei-Abreu Goodger, Anton Enright and Elena Vigorito.
The Laboratory for Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK; The Wellcome trust Sanger Institute, Cambridge, UK.

MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by repressing translation or by inducing mRNA degradation. Dicer, an enzyme responsible for miRNA biogenesis is required for T-cell function suggesting regulatory roles for miRNAs in lymphocytes. However, specific roles for individual miRNAs are only just beginning to emerge. MiR-155 is encoded within an exon of the non-coding RNA known as bic and high levels of bic expression are induced upon antigen receptor stimulation of B and T cells, as well as TLR stimulation macrophages and dendritic cells. High levels of bic/miR-155 are found in B cell lymphomas and solid tumors, indicating that this locus may also be linked to cancer. Indeed, transgenic miR-155 mice overexpressing miR155 develop B cell malignancies.
To define the in vivo role of bic/miR-155 (bic) we have generated bic-deficient mice. These mice are immunodeficient and fail to generate high levels of class-switched antibody upon immunization with thymus-dependent and thymus independent antigens. This defect is intrinsic to B cells and manifested at the level of differentiation of switched plasmablasts into mature antibody secreting plasma cells. In addition, bic-deficient T cells are defective in the differentiation into Th1 and Th2 lineages. Microarray analysis of bic–deficient B and T cells under different conditions has revealed the spectrum of targets regulated by a miRNA-155 and suggested mechanisms for the regulation of lymphocyte differentiation by a single miRNA.

ULS labeling of miRNA is strongly down-scalable and compatible with many platforms
Erik Jan Klok, Ph.D., Product Development Manager Microarrays
Kreatech Biotechnology B.V., Amsterdam, The Netherlands

ULS-Cy3/Cy5 is a chemical labeling technology that allows down-scaling of the labeling reaction to any small amount of miRNA; if desired, it is possible to label an amount of 25 ng of total RNA. Furthermore, ULS-labeled miRNA is compatible with all miRNA microarray platforms that we have tested so far including those from Agilent, Exiqon, Invitrogen and CombiMatrix showing optimal sensitivity and specificity.
ULS technology allows labeling of RNA molecules independent of their size, and is therefore very suitable for miRNA labeling. ULS labels the miRNA molecules directly in their natural form, without enzymes or linkers.

MicroRNA Expression Profiling and Functional Analysis of Specific MicroRNAs in Colorectal Cancer
Greg M. Arndt1, Lesley Dossey2, Lara M. Cullen1, Angela Lai1, Riki Druker1, Michael Eisbacher1, Chunyan Zhang1, Nham Tran1, Hongtao Fan2, Kathy Retzlaff2, Jay Nelson3, Anton Bittner3, and Mitch Raponi2.
1. Johnson & Johnson Research Pty Ltd. 2. Ortho-Clinical Diagnostics. 3. Johnson & Johnson Pharmaceutical Research & Development LLC.

Presentation by Greg M. Arndt, PhD. Research Director, Target Discovery, Johnson & Johnson Research Pty Limited, Sydney, NSW 1430, AUSTRALIA.

MicroRNAs (miRNA) are short non-coding RNAs that control the expression of multiple proteins through various mechanisms. To date, there have been 474 human miRNAs identified and, using computational algorithms, it is predicted that over 1000 miRNA genes may exist in mammalian cells. Using expression profiling, it has been shown that miRNAs are differentially expressed in different cancers and limited functional studies have implicated specific miRNAs as either oncogenes or tumor suppressors. In this talk, I will review expression profiling of human miRNAs, and in vitro and in vivo functional studies of selected miRNAs, in colorectal cancer.

MicroRNA profiles in Cerebral Ischemia: Key players in post-ischemic gene regulation
Raghu Vemuganti, PhD. Assistant Professor, Dept of Neurological Surgery, University of Wisconsin, Madison WI 53792, USA

A. Dharap and R.Vemuganti
Dept of Neurological Surgery and Neuroscience Training Program, University of Wisconsin, Madison, WI, USA

By virtue of their position between genomic DNA and functional proteins, mRNAs are considered the key controllers of cellular functions. Several recent studies have shown significantly altered mRNA profiles following focal ischemia, which play a role in promoting the ischemic pathophysiology. The mRNA transcription and translation of proteins involves several factors including transcription factors, RNAses, ribosomes and other protein synthesis machinery. Recent studies have shown that in addition to the conventional mechanisms, mRNAs are finely controlled by a group of evolutionarily well-conserved, 18 to 26 nucleotide long, non-coding RNAs known as microRNAs (miRNAs). Although the mechanism of their action is not completely elucidated, miRNAs are though to modulate mRNA function either by degrading them and/or by inhibiting their translation. Studying the expression and the function of miRNAs following brain injury will complement the knowledge on altered mRNA function and provide improved therapeutic targets for preventing neuronal death. Hence, we conducted miRNA microarray profiling in adult rat cerebral cortex as a function of reperfusion time following transient middle cerebral artery occlusion (MCAO). In normal rat brains, of the 238 miRNAs studied, 99 were expressed at a moderate to high level (range: 2,000 to 52,512 units). By 3h of reperfusion, 3 miRNAs were upregulated and 9 miRNAs were down-regulated. The number of miRNAs that showed altered levels increased progressively such that by 72h reperfusion, 36 were up- and 33 were down-regulated. At least 30 miRNAs showed >7 fold change at various reperfusion time points compared to sham control. Using bioinformatics tools, we could correlate decreased miRNAs to many mRNAs known to be increased after focal ischemia. This indicates that miRNAs might play a crucial role in the mRNA expression and the post-ischemic brain damage.

Control of coleopteran insect pests through RNA interference
Jim Roberts, Ph.D. Gene Suppression Team Lead, Monsanto, St. Louis, MO, USA

Small interfering RNAs (siRNAs) function in important regulatory roles in plants and animals. Double stranded RNA (dsRNA) has been widely exploited as a precursor to generate siRNA to suppress endogenous genes, whereas there are far fewer examples using dsRNA directed to exogenous RNA. We will present studies that explore the use of dsRNA to control major insect pests in agriculture. Western corn rootworm, (WCR, Diabrotica virgifera virgifera) larvae exhibit significant stunting and mortality in an artificial diet bioassay assay when fed a diet containing dsRNAs derived from a variety of WCR genes. Similar results were obtained with two other coleopteran species, Diabrotica undecimpunctata howardii (southern corn rootworm, SCR) and Leptinotarsa decemlineata (Colorado potato beetle, CPB). Analysis of treated WCR larvae demonstrated an early and specific decrease in the levels of mRNA corresponding to the WCR gene target. Transgenic corn plants engineered to express dsRNA containing WCR sequences show significant reduction in WCR feeding damage in a growth chamber assay. This is the first reported exploitation of the RNAi pathway to control an insect pest via in planta expression of a dsRNA.

MicroRNA Expression in a Carcinogen-Induced Cancer Model
Yan Zeng, Ph.D. Assistant Professor of Pharmacology, Department of Pharmacology and Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA

Zeng, Y., Kalscheuer, S., Zhang, X., Upadhyaya, P., and Hecht, S.
Department of Pharmacology and Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA

A number of studies had examined the microRNA (miRNA) expression profile in human lung cancer and identified dozens of miRNAs with increased or decreased expression levels in cancerous tissues compared with normal tissues. In addition, the expression of several miRNAs had been correlated with lung cancer diagnosis and prognosis, further underscoring the importance of miRNAs to the pathogenesis of human disease. The function of these miRNAs and how they contribute to the development of human lung cancer, however, is largely unknown.

We have initiated a study that uses a carcinogen-induced cancer model to investigate the involvement of miRNAs in cancer development. NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), a chemical present in tobacco products, causes cancers in humans and in
laboratory animals such as rats and mice. We have determined the miRNA expression profiles in the lungs of rats treated with NNK up to twenty weeks (still in the early stages of tumorigenesis). Our results show that a small number of miRNAs alter their expression levels upon NNK treatment, as measured by miRNA microarrays and confirmed by Northern blot analyses. Significantly, these miRNAs are a subset of miRNAs that had been previously reported to change in human lung cancer. Such miRNAs may, therefore, represent early-response miRNAs that signify the molecular changes associated with carcinogenesis in the lung. We are currently studying the function of these miRNAs as well as miRNA expression profiles in rats treated with NNK for longer than 20 weeks.

RNA-mediated induction of hereditary epigenetic modification
Minoo Rassoulzadegan, PhD. Unité 636 de l’INSERM, Université de Nice-Sophia Antipolis, 06108 Nice cedex, France

Minoo Rassoulzadegan1, Valérie Grandjean1, Kay Wagner1, Hossein Ghanbarian1, Nicole Wagner1, Pierre Gounon2, and François Cuzin1
1 Unité 636 de l’INSERM, Université de Nice-Sophia Antipolis, 06108 Nice cedex, France.
2 Centre Commun de Microscopie Appliquée, Université de Nice-Sophia Antipolis, 06108 Nice cedex, France

Known as paramutation and extensively studied in plants, hereditary epigenetic variation was only recently reported in the mouse. We have reported a modification in the phenotypic expression of the wild type allele of the Kit receptor gene in the progeny of heterozygotes with a null insertion mutant. In spite of a wild type genomic structure, the modified homozygotes maintain the “White Spotted” phenotype characteristic of Kit mutants, in this case a white tip of the tail and white feet. This epigenetic modification is efficiently inherited in the absence of the mutant allele. a modification of the Kit locus resulting in the transgenerational maintenance of a mutant phenotype with a wild type genotype. RNA-mediated inheritance, suggested by accumulation of RNA in sperm, was further supported by the efficient establishment of the epigenetic state after microinjection in one-cell embryos of RNA. Concept of RNA mediated epigenetic determination was then extended to hereditary heart hypertrophy, by microinjection in early embryos of miR‑1, a muscle cells specific microRNA. As it is, the mouse paramutation may provide a model for human familial diseases, including cardiomyopathies, whose inheritance is not fully explained in Mendelian terms.

Hepatitis C virus replication depends on liverspecific microRNA miR-122
Sylvia Schultz, PhD. Postdoctoral Fellow in the Lab of Professor Peter Sarnow
Dept. Of Microbiology and Immunology, Stanford University Medical School, Stanford, CA 94305, USA

Sylvia Schuetz1, Catherine Jopling2, Kara Norman1, Peter Sarnow1
1Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA; 2Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK

MiR-122 is the most abundant microRNA in liver cells. We have discovered that miR-122 interacts with the 5’NCR of the HCV genome and that this interaction supports viral replication. MiR-122 does not affect viral mRNA translation or stability, pointing towards a function of miR-122 in viral replication. Luciferase constructs with HCV-miR-122 binding sites show that this site acts like a regular microRNA-site when present in the 3’UTR, but not in the 5’ UTR, of the construct. These results show an unprecedented upregulation of a target RNA by a microRNA and suggest miR-122 to be a potential target for antiviral therapy.

Discovery and expression profiling of small RNAs by deep sequencing
Eugene Berezikov, PhD. Group Leader, Hubrecht Institute, Utrecht, The Netherlands; and Co-founder, InteRNA Genomics, Bilthoven, The Netherlands.

Advances in next generation sequencing technology have boosted the area of small RNA research. Massively parallel sequencing of the small RNA complement of cells, tissues and patient samples provides an unbiased approach for the simultaneous discovery and detection of novel and known small RNAs including microRNAs. Although experimental deep sequencing approaches are maturing fast, analysis of the resulting data requires advanced algorithms, state-of-the-art infrastructure and highly skilled bioinformaticians for processing. Informatics thus becomes a bottleneck for many research laboratories in using deep sequencing technologies. We have developed a modular pipeline for the analysis and interpretation of high-throughput small RNA sequencing data. Output formats include for example summary tables and text files, fasta sequence files, frequency and heat maps, homology and orthology information, tracks on genome browsers, etc. Analysis of several datasets generated on 454/Roche and Solexa/Illumina platforms will be presented.

A microarray platform for miRNA profiling that is both sensitive and robust.
Petula D’Andrade, PhD. Agilent Product R&D Labs Specialist, Agilent Technologies

Agilent’s miRNA profiling system utilizes a simple direct miRNA labeling strategy using 100 ng of total RNA without size fractionation. This labeling strategy, in conjunction with our probe design, provides a sensitive and specific miRNA microarray assay that is capable of over 4 orders of magnitude in measurement linear dynamic range. In addition, the Agilent miRNA platform takes advantage of several data extraction steps unique to the miRNA platform, which enhance and monitor the quality of our measurements. Data will be presented that demonstrate the reproducibility, sensitivity, and dynamic range of the platform. Through the combination of Agilent’s high quality DNA microarrays and the design and processing attributes unique to the miRNA platform, we are able to offer a robust and sensitive platform for miRNA profiling.

Non-conserved intronic motifs are related to piRNAs and linked to a conserved set of functions in human and mouse
Aristotelis Tsirigos, PhD. Bioinformatics & Pattern Discovery Group, Computational Biology Center, IBM Thomas J Watson Research Center
Yorktown Heights, NY, USA

We report that frequent intronic motifs, termed pyknons, are present outside human-mouse alignments and known repeat regions, and account for 9% of the previously uncharacterized intronic space. We also demonstrate that, in the absence of underlying sequence conservation, intronic pyknon instances are associated with the same set of biological processes and molecular functions in both the human and mouse genomes. Our results implicate a substantial fraction of intronic regions in functional links that are conserved in human and mouse; however, the underlying sequences are organism-specific and thus, not conserved between the two genomes.

Discovery of microRNA cancer biomarkers - using miRCURY™ LNA Array
Carsten Alsbo, PhD.,Product Manager. Exiqon A/S, Vedbaek, Denmark

The miRCURY™ LNA product line is unique for detection, profiling and inhibition of microRNAs. This presentation will give a brief description of the miRCURY™ LNA product line and will focus on a study on the global expression profiles of microRNAs in breast cancer. We used the miRCURY™ LNA Array platform for analysis of microRNA expression patterns from tumor and normal breast tissue, and we have identified several novel microRNA candidates as well as microRNA previously reported to be associated with breast cancer. Some of these novel microRNA signatures could have diagnostic and prognostic potential for breast cancer patients.

Affinity isolation of small RNAs with the plant viral protein p19
Larry A. McReynolds, PhD., Director of RNA Biology, New England Biolabs, Ipswich, MA, USA

Jingmin Jin, Catherine Poole, Meghan Therrrien, Imane Azzouzi, Larry McReynolds
Department of RNA Biology, New England Biolabs, Ipswich, MA

The p19 from the plant Carnation Italian Ringspot Virus binds siRNAs with high affinity [1,2,3]. We developed p19 as an affinity reagent for binding endogenous dsRNA. A p19 fusion was constructed with an N -terminal maltose binding protein [MBP] and a C terminal chitin binding domain [CBD] that retained its binding affinity for siRNA.  The presence of the MBP moiety permitted purification of the fusion protein at high yields using amylose resin.

The p19 fusion binds siRNAs in a size dependent sequence independent manner.   This was demonstrated using a competitive gel shift assay to determine the relative binding affinities of the p19 fusion protein to selected oligonucleotides.  It was determined that p19 preferentially binds 21 bps siRNAs compared with 17 bp or 25 bp siRNAs and prefers 21 siRNAs over 21 bp miRNAs which contain mismatched base pairs.  In addition p19 fusion binds to RNA:DNA hybrids but not DNA; DNA, ssRNA or ribosomal RNA.

Fluorescence polarization using FAM labeled siRNA was used to monitor the kinetics of binding. Small endogenous dsRNAs were isolated from total RNA extracts of C. elegans and from the parastic nematode Dirofilaria immitis by immobilization on chitin columns after binding to p19 fusion protein.  The isolated endogenous dsRNA represents less then 0.005% of the total cytoplasmic RNA. The mobility of the endogenous dsRNA correlates with a size of about 23-25 bp.  This larger size may be explained by the fact that these molecules are secondary siRNAs that have a 5’ triphosphate. Additional studies are needed to further characterize these endogenous RNAs.  The p19 fusion protein is a useful reagent for the discovery of siRNAs of unknown sequence based on their structure not just their electrophoretic mobility.

References:
[1] Silhavy, D et al, EMBO J 21:3070-80 (2002)
[2]  Vargason, JM e al, Cell 115:799-811 (2003)
[3] Baulcombe DC and Molnar A, Trends Biochem. Sci. 29:279-81 (2004)

The role of microRNAs in cartilage development
Tamas Dalmay, PhD. Senior Lecturer in Cell and Molecular Biology, School of Biological Sciences, University of East Anglia, Norwich, UK

MiRNA-140 was shown to be specifically expressed in cartilage tissue of zebrafish embryos. We analysed its expression pattern in mouse embryos and found miR140 accumulation in every cartilaginous tissue of the developing mouse embryo. Predicted targets were tested using the luciferase assay system and several of them were validated. Histone deacetylase 4 down-regulation by miR140 was further confirmed by Western blot. Recently, we initiated experimental target identification and analysed mRNA expression followed by over-expression or down-regulation of miR140 in a chondrocyte cell line.