Nielsen H. RNA: Methods and Protocols

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Contents

Preface .......................................... v

Contributors ....................................... xiii

Electronic Supplementary Material 1

Electronic Supplementary Material 2

PART I: RNA METHODS

1. The Transcriptional Landscape ........................... 3

Henrik Nielsen

2. Working with RNA

................................. 15

Henrik Nielsen

3. Synthesis of RNA by In Vitro Transcription

.................... 29

Bertrand Beckert and Benoît Masquida

4. Efﬁcient Poly(A)

RNA Selection Using LNA Oligo(T) Capture ......... 43

Nana Jacobsen, Jens Eriksen, and Peter Stein Nielsen

5. Genome Browsers

................................. 53

Elfar Torarinsson

6. Web-Based Tools for Studying RNA Structure and Function

........... 67

Ajish D. George and Scott A. Tenenbaum

7. Northern Blotting Analysis

............................. 87

Knud Josefsen and Henrik Nielsen

8. Rapid Ampliﬁcation of cDNA Ends (RACE)

.................... 107

Oladapo Yeku and Michael A. Frohman

9. Fractionation of mRNA Based on the Length of the Poly(A) Tail

......... 123

Hedda A. Meijer and Cornelia H. de Moor

10. Analysis of Mutations that Inﬂuence Pre-mRNA Splicing

............. 137

Zhaiyi Zhang and Stefan Stamm

11. S1 Nuclease Analysis of Alternatively Spliced mRNA

................ 161

Martin Lützelberger and Jørgen Kjems

12. Promises and Challenges in Developing RNAi as a Research Tool and Therapy

.. 173

Mouldy Sioud

xii Contents

13. Inhibition of Gene Function in Mammalian Cells Using Short-Hairpin

RNA (shRNA)

................................... 189

Jørn Remi Henriksen, Jochen Buechner, Cecilie Løkke,

Trond Flægstad, and Christer Einvik

14. Validation of RNAi by Real Time PCR

....................... 205

Knud Josefsen and Ying C. Lee

15. Proﬁling RNA Polymerase II Using the Fast Chromatin

Immunoprecipitation Method

........................... 219

Joel Nelson, Oleg Denisenko, and Karol Bomsztyk

PART II: RNP METHODS

16. The Post-transcriptional Operon .......................... 237

Scott A. Tenenbaum, Jan Christiansen, and Henrik Nielsen

17. RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-

Microarray (Chip) Proﬁling

............................. 247

Ritu Jain, Tiffany Devine, Ajish D. George, Sridar V. Chittur,

Timothy E. Baroni, Luiz O. Penalva, and Scott A. Tenenbaum

18. Isolation of RNP Granules

............................. 265

Lars Jønson, Finn Cilius Nielsen, and Jan Christiansen

19. Electrophoretic Mobility Shift Assay for Characterizing RNA–Protein

Interaction

..................................... 275

Keith T. Gagnon and E. Stuart Maxwell

20. Polysome Analysis and RNA Puriﬁcation from Sucrose Gradients

......... 293

Tomáš Mašek, Leoš Valášek and Martin Pospíšek

21. Prediction of Targets for MicroRNAs

....................... 311

Morten Lindow

22. Outsourcing of Experimental Work

........................ 319

Henrik Nielsen

Subject Index

....................................... 327

Contributors

T IMOTHY E. BARONI • Department of Biomedical Sciences, School of Public Health,

Gen

∗

Sis Center for Excellence in Cancer Genomics, University at Albany-SUNY,

Rensselaer, NY, USA

ERTRAND BECKERT • Department of Cellular and Molecular Medicine, The Panum

Institute, University of Copenhagen, Copenhagen, Denmark

AROL BOMSZTYK • Department of Pharmacology, School of Medicine, University of

Washington, Seattle, WA, USA

OCHEN BUECHNER • Department of Pediatrics, University Hospital of North-Norway,

Tromsø, Norway

RIDAR V. CHITTUR • Department of Biomedical Sciences, School of Public Health,

Gen

∗

Sis Center for Excellence in Cancer Genomics, University at Albany-SUNY,

Rensselaer, NY, USA

AN CHRISTIANSEN • Department of Biology, University of Copenhagen, Copenhagen,

Denmark

ORNELIA H. DE MOOR • RNA Biology Group, School of Pharmacy, Centre for Biomolec-

ular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

LEG DENISENKO • University of Washington Medicine at Lake Union, University of

Washington, Seattle, WA, USA

IFFANY DEVINE • Department of Biomedical Sciences, School of Public Health,

Gen

∗

Sis Center for Excellence in Cancer Genomics, University at Albany-SUNY,

Rensselaer, NY, USA

HRISTER EINVIK • Department of Pediatrics, University Hospital of North-Norway,

Tromsø, Norway

ENS ERIKSEN • Laboratory of Oncology, Herlev University Hospital, Herlev, Denmark

ROND FLÆGSTAD • Department of Pediatrics, University Hospital of North-Norway,

Tromsø, Norway; Department of Pediatrics, Institute of Clinical Medicine, University of

Tromsø, Tromsø, Norway

ICHAEL A. FROHMAN • Department of Pharmacology, Center for Developmental

Genetics, Stony Brook University, Stony Brook, NY; Center for Molecular Medicine, Stony

Brook University, Stony Brook, NY, USA

EITH T. GAGNON • Department of Pharmacology, University of Texas Southwestern

Medical Center, Dallas, TX, USA

JISH D. GEORGE • Department of Biomedical Sciences, School of Public Health,

Gen

∗

Sis Center for Excellence in Cancer Genomics, University at Albany-SUNY,

Rensselaer, NY, USA

ØRN REMI HENRIKSEN • Department of Pediatrics, University Hospital of North-

Norway, Tromsø, Norway

ANA JACOBSEN • Exiqon, Vedbaek, Denmark

ITU JAIN • Department of Biomedical Sciences, School of Public Health, Gen

∗

Sis

Center for Excellence in Cancer Genomics, University at Albany-SUNY, Rensselaer, NY,

USA

xiii

xiv Contributors

LARS JØNSON • Department of Clinical Biochemistry, Copenhagen University Hospital,

Copenhagen, Denmark

NUD JOSEFSEN • The Bartholin Institute, Copenhagen University Hospital, Copenhagen,

Denmark

ØRGEN KJEMS • Department of Molecular Biology, University of Århus, Århus C,

Denmark

ING C. LEE • Cellular and Metabolic Research Section, Biomedical Institute, University

of Copenhagen, Copenhagen, Denmark

ORTEN LINDOW • Santaris Pharma A/S, Hørsholm, Denmark

ECILIE LØKKE • Department of Pediatrics, Institute of Clinical Medicine, University of

Tromsø, Tromsø, Norway

ARTIN LÜTZELBERGER • Institute of Genetics, Technical University of Braunschweig,

Braunschweig, Germany

OMÁŠ MAŠEK • Department of Genetics and Microbiology, Charles University in Prague,

Prague, Czech Republic

ENOÎT MASQUIDA • Architecture et Réactivité de l’ARN, Université de Strasbourg,

CNRS, IBMC, Strasbourg, France

E. S

TUART MAXWELL • Department of Molecular and Structural Biochemistry, North

Carolina State University, Raleigh, NC, USA

EDDA A. MEIJER • Toxicology unit, Medical Research Council, Hodgkin Building,

University of Leicester, Lancaster Road, Leicester LE, 9HN, UK

OEL NELSON • Molecular and Cellular Biology Program, University of Washington,

Seattle, WA, USA

INN CILIUS NIELSEN • Department of Clinical Biochemistry, Copenhagen University

Hospital, Copenhagen, Denmark

ENRIK NIELSEN • Department of Cellular and Molecular Medicine, The Panum

Institute, University of Copenhagen, Copenhagen, Denmark

ETER STEIN NIELSEN • Exiqon, Vedbaek, Denmark

UIZ O. PENALVA • Department of Cellular and Structural Biology, Children’s Cancer

Research Institute, San Antonio, TX, USA

ARTIN POSPÍŠEK • Department of Genetics and Microbiology, Faculty of Science, Charles

University in Prague, Prague, Czech Republic

OULDY SIOUD • Molecular Medicine Group, Department of Immunology, Institute for

Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway

TEFAN STA MM • Department of Molecular and Cellular Biochemistry, Biomedical

Biological Sciences Research Building, College of Medicine, University of Kentucky,

Lexington, KY, USA

COTT A. TENENBAUM • College of Nanoscale Science and Engineering, Nanoscale

Constellation, University at Albany-SUNY, Rensselaer, NY, USA

LFAR TORARINSSON • Division of Genetics and Bioinformatics, Department of

Basic Animal and Veterinary Science, University of Copenhagen, Frederiksberg C,

Denmark; Department of Natural Sciences, Faculty of Life Sciences, University of

Copenhagen, Frederiksberg C, Denmark

EOŠ VALÁŠEK • Laboratory of Regulation of Gene Expression, Institute of Microbiology,

Academy of Sciences of the Czech Republic, Prague, Czech Republic

Contributors xv

OLADAPO YEKU • Department of Pharmacology, Center for Developmental Genetics,

Stony Brook University, Stony Brook, NY, USA

HAIYI ZHANG • Department of Molecular and Cellular Biochemistry, Biomedical

Biological Sciences Research Building, College of Medicine, University of Kentucky,

Lexington, KY, USA

Part I

RNA Methods

Chapter 1

The Transcriptional Landscape

Henrik Nielsen

Abstract

The application of new and less biased methods to study the transcriptional output from genomes, such

as tiling arr ays and deep sequencing, has revealed that most of the genome is transcribed and that there

is substantial overlap of transcripts derived fr om the two strands of DNA. In protein coding regions, the

map of transcripts is very complex due to small transcripts from the ﬂanking ends of the transcription unit,

the use of multiple start and stop sites for the main transcript, production of multiple functional RNA

molecules from the same primary transcript, and RNA molecules made by independent transcription

from within the unit. In genomic regions separating those that encode proteins or highly abundant RNA

molecules with known function, transcripts are generally of low abundance and short-lived. In most of

these cases, it is unclear to what extent a function is related to transcription per se or to the RNA products.

Key words: Pervasive transcription, promoter, ncRNA, antisense.

1. Introduction

Genetic information is stored in DNA and is expressed through

copying into RNA molecules by the action of RNA polymerase

in a process known as transcription. The RNA molecules (tran-

scripts) can either be the ﬁnal product themselves (e.g., ribosomal

RNA and transfer RNA) or be messenger RNA u sed to instruct

the synthesis of protein molecules that in these cases are the ulti-

mate products of gene expression. Together the RNA molecules

transcribed from a genome are referred to as the transcriptome.

In the traditional view of the transcriptional landscape, i.e., the

map of transcripts derived from the genome, the transcripts are

made fr om well-deﬁned transcription units with discrete bound-

aries scattered over the genome. In some organisms (typical of

H. Nielsen (ed.), RNA, Methods in Molecular Biology 703,