M e t h o d s i n M o l e c u l a r B i o l o g y ™

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

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MicroRNAs and the Immune SystemMethods and Protocols

Edited by

Silvia Monticelli

Institute for Research in Biomedicine, Bellinzona, Switzerland

EditorSilvia MonticelliInstitute for Research in BiomedicineBellinzonaSwitzerlandsilvia.monticelli@irb.unisi.ch

ISSN 1064-3745 e-ISSN 1940-6029ISBN 978-1-60761-810-2 e-ISBN 978-1-60761-811-9DOI 10.1007/978-1-60761-811-9

Library of Congress Control Number: 2010935486

© Springer Science+Business Media, LLC 2010All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

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Preface

The hematopoietic system is a paradigm for the differentiation of distinct cell lineages from multipotent progenitors. Differentiation is modulated by an intricate network of growth and transcription factors that simultaneously regulate the commitment, prolifera-tion, apoptosis, and maturation of hematopoietic stem and progenitor cells. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression by bind-ing to target messenger RNAs and inducing translational repression, cleavage, or destabi-lization of the target. Each miRNA can potentially regulate expression of a distinct set of genes and therefore miRNAs appear ideally suited to rapidly adjusting protein concentra-tions in cells, as would be expected to be required during cell differentiation. In fact, certain miRNAs are differentially expressed, both spatially and temporally, in many types of immune cells. Moreover, consistent with the discovery that miRNAs modulate gene expression, altered miRNA expression has been associated with various types of diseases, including cancer.

The overall importance of miRNAs during hematopoiesis has been investigated by specific disruption of steps in miRNA biogenesis, indicating a critical function for miRNAs in the biology of cells that constitute the immune system. In this volume of Methods in Molecular Biology, various methods to study miRNA expression in cells of the immune system are described, such as splinted ligation and qRT-PCR assays, as well as high-throughput profiling through cloning, deep sequencing and microarrays. A complemen-tary approach to expression profiling is the use of miRNA reporter vectors for assaying miRNA activity. Moreover, a method to visualize miRNAs in situ in bone marrow cells is described. Besides providing an overview for the most up-to-date techniques to study miRNA expression, this book encompasses methods to study miRNA functions in various cell types of the immune system, using loss- and gain-of-function techniques, both at a single cell-type level and in entire model organisms, as well as for studies of miRNAs in the context of viruses and the immune response.

One of the most elusive areas in the miRNA field is target recognition. As a result, different computational approaches have been developed to predict miRNA target sites throughout the transcriptome. Here, tools are also provided to help understanding and navigating these bioinformatics databases. Besides the analysis of miRNA expression and function, a major challenge is represented by the precise understanding of miRNA func-tion at a molecular level. We therefore, provide protocols for the emerging field of miR-NAs posttranscriptional modifications (i.e., RNA editing), as well as for NMR structures of miRNA:mRNA complexes.

This volume of Methods will be of interest to immunologists approaching the study of miRNAs in cells of the immune system, biochemists and molecular biologists interested in the exploration of the posttranscriptional modifications and mechanisms of action of miRNAs, as well as to virologists and bioinformaticians.

Dr. Silvia Monticelli

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vii

Acknowledgments

I would like to especially thank Dr. Luisa Granziero for the invaluable help and support throughout the preparation of this volume.

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vAcknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiContributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Part I analysIs of mIrna ExPrEssIon: ClassIC mEthods rEvIsItEd

1. A Rapid, Quantitative Assay for Direct Detection of MicroRNAs and Other Small RNAs Using Splinted Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . 3Sangpen Chamnongpol, Patricia A. Maroney, and Timothy W. Nilsen

2. Normalization of MicroRNA Quantitative RT-PCR Data in Reduced Scale Experimental Designs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Gary J. Latham

3. MicroRNA Detection in Bone Marrow Cells by LNA-FISH . . . . . . . . . . . . . . . . . 33Silvana Debernardi and Amanda Dixon-McIver

4. Measuring MicroRNA Expression in Size-Limited FACS-Sorted and Microdissected Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Kai P. Hoefig and Vigo Heissmeyer

Part II hIgh-throughPut analysIs of mIrnas

5. MicroRNA Cloning from Cells of the Immune System . . . . . . . . . . . . . . . . . . . . . 67Haoquan Wu, Joel Neilson, and N. Manjunath

6. High-Throughput Profiling in the Hematopoietic System. . . . . . . . . . . . . . . . . . . 79Muller Fabbri, Riccardo Spizzo, and George A. Calin

7. Construction of Small RNA cDNA Libraries for Deep Sequencing . . . . . . . . . . . . 93Molly F. Thomas and K. Mark Ansel

8. MicroRNA-Profiling in Formalin-Fixed Paraffin-Embedded Specimens. . . . . . . . . 113Ulrich Lehmann

Part III funCtIonal analysIs of mIrnas In thE ImmunE systEm: gaIn-of-funCtIon

9. Expression of miRNAs in Lymphocytes: A Review . . . . . . . . . . . . . . . . . . . . . . . . 129Raquel Malumbres and Izidore S. Lossos

10. Mouse Models for miRNA Expression: The ROSA26 Locus . . . . . . . . . . . . . . . . . 145Stefano Casola

11. Regulation of Monocytopoiesis by MicroRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . 165Laura Fontana, Antonio Sorrentino, and Cesare Peschle

12. MicroRNA Activity in B Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177Virginia G. de Yébenes and Almudena R. Ramiro

13. Isolation and Characterization of MicroRNAs of Human Mature Erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Carolyn Sangokoya, Gregory LaMonte, and Jen-Tsan Chi

x Contents

14. Stable Overexpression of miRNAs in Bone Marrow-Derived Murine Mast Cells Using Lentiviral Expression Vectors . . . . . . . . . . . . . . . . . . . . 205Ramon J. Mayoral and Silvia Monticelli

15. Monitoring MicroRNA Activity and Validating MicroRNA Targets by Reporter-Based Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215Alessia Baccarini and Brian D. Brown

Part Iv funCtIonal analysIs of mIrnas In thE ImmunE systEm: loss-of-funCtIon

16. Lentivirus-Mediated Antagomir Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237Ewa Surdziel, Matthias Eder, and Michaela Scherr

Part v mIrna Post-transCrIPtIonal modIfICatIons and mEChanIsms of aCtIon

17. Solution Structure of miRNA:mRNA Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . 251Mirko Cevec and Janez Plavec

18. MiRNA Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Dylan E. Dupuis and Stefan Maas

Part vI BIoInformatIC analysIs and targEt PrEdICtIon

19. Computational Prediction of MicroRNA Targets . . . . . . . . . . . . . . . . . . . . . . . . . 283Xiaowei Wang

20. Large-Scale Integration of MicroRNA and Gene Expression Data for Identification of Enriched MicroRNA–mRNA Associations in Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297Preethi H. Gunaratne, Chad J. Creighton, Michael Watson, and Jayantha B. Tennakoon

Part vII mIrna and vIrusEs

21. Identification and Validation of the Cellular Targets of Virus-Encoded MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Kin-Hang Kok, Ting Lei, and Dong-Yan Jin

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

xi

Contributors

K. MarK ansel • Department of Microbiology & Immunology, Strategic Asthma Basic Research Center, University of California San Francisco, San Francisco CA, USA

alEssIa BaCCarInI  •  Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York NY, USA

BrIan D. Brown  •  Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York NY, USA

GeorGe Calin • Department of Experimental Therapeutics and Department of Cancer Genetics, University of Texas M .D . Anderson Cancer Center, Houston TX, USA

stefano Casola • IFOM, The FIRC Institute of Molecular Oncology Foundation, Milan, Italy

MirKo CeveC • Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia

sanGpen ChaMnonGpol • Affymetrix, Inc, Cleveland, OH, USAJen-tsan Chi • Department of Molecular Genetics and Microbiology, The Institute

for Genome Sciences and Policy, Duke University School of Medicine, Durham NC, USA

Chad J. CreiGhton • Dan Duncan Cancer Center, Baylor College of Medicine, Houston TX, USA

virGinia G. de Yébenes • DNA Hypermutation and Cancer Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain

silvana debernardi • Medical Oncology Centre, Barts & The London School of Medicine and Dentistry, Institute of Cancer, Queen Mary University of London, London, UK

aManda dixon-MCiver • Medical Oncology Centre, Barts & The London School of Medicine and Dentistry, Institute of Cancer, Queen Mary University of London, London, UK

dYlan e. dupuis • Department of Biological Sciences, Lehigh University, Bethlehem PA, USA

Matthias eder • Medizinische Hochschule Hannover, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany

Muller fabbri • Department of Molecular Virology, Immunology and Medical Genetics, the Ohio State University, Columbus, OH, USA

laura fontana • Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

preethi h. Gunaratne • Department of Biology & Biochemistry, University of Houston, HoustonTX, USA Department of Pathology, University of Houston, Houston TX, USA Human Genome Sequencing Center, University of Houston, Houston TX, USA

VIgo HEIssmEyEr  •  Helmholtz Center Munich, Institute of Molecular Immunology, Munich, Germany

xii Contributors

Kai p. hoefiG • Helmholtz Center Munich, Institute of Molecular Immunology, Munich, Germany

donG-Yan Jin • Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong

Kin-hanG KoK • Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong

GreGorY laMonte • Department of Molecular Genetics and Microbiology, The Institute for Genome Sciences and Policy, Duke University School of Medicine, Durham, NC, USA

GarY J. lathaM • Asuragen, Inc, AustinTX, USAulriCh lehMann • Institute of Pathology, Medizinische Hochschule Hannover,

Hannover, Germany tinG lei • Department of Biochemistry, The University of Hong Kong,

Pokfulam, Hong Kong izidore s. lossos • Division of Hematology-Oncology and Molecular and Cellular

Pharmacology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami FL, USA

stefan Maas • Department of Biological Sciences, Lehigh University, Bethlehem PA, USA raquel MaluMbres • Department of Oncology, Center for Applied Medical

Research, PamplonaNavarra, Spainn. ManJunath • Department of Biomedical Sciences, Center of Excellence

in Infectious Disease Research, Paul L . Foster School of Medicine, Texas Tech University Health Sciences Center, El PasoTX, USA

patriCia a. MaroneY • Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, ClevelandOH, USA

raMon J. MaYoral • Institute for Research in Biomedicine, Bellinzona, Switzerlandsilvia MontiCelli • Institute for Research in Biomedicine, Bellinzona, SwitzerlandJoel neilson • Department of Molecular Physiology and Biophysics, Baylor College

of Medicine, Houston TX, USAtiMothY W. nielsen • Center for RNA Molecular Biology and Department

of Biochemistry, Case Western Reserve University, Cleveland OH, USACesare pesChle • IRCCS Multimedica, Milan, ItalyJanez plaveC • Slovenian NMR Centre, National Institute of Chemistry,

Ljubljana, Slovenia alMudena r. raMiro • DNA Hypermutation and Cancer Group,

Spanish National Cancer Research Center (CNIO), Madrid, SpainCarolYn sanGoKoYa • Department of Molecular Genetics and Microbiology,

The Institute for Genome Sciences and Policy, Duke University School of Medicine, DurhamNC, USA

MiChaela sCherr • Medizinische Hochschule Hannover, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany

antonio sorrentino • Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

riCCardo spizzo • Department of Experimental Therapeutics and Department of Cancer Genetics, The University of Texas M .D . Anderson Cancer Center, Houston TX, USA

xiiiContributors

eWa surdziel • Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Medizinische Hochschule Hannover, Hannover, Germany

JaYantha b. tennaKoon • Department of Biology & Biochemistry, University of Houston, Houston TX, USA

MollY f. thoMas • Department of Microbiology & Immunology, Strategic Asthma Basic Research Center, University of California San Francisco, San Francisco CA, USA

xiaoWei WanG • Department of Radiation Oncology, Washington University School of Medicine, St . Louis MO, USA

MiChael Watson • Bioinformatics Group, Institute for Animal Health, Compton, UK

haoquan Wu • Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L . Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso TX, USA