High-Throughput RNAi Screening: Methods and Protocols

275 Pages·2016·9.18 MB·English

by  David O. Azorsa

Methods in
Molecular Biology 1470
David O. Azorsa
Shilpi Arora Editors
High-Throughput
RNAi Screening
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB , UK
For further volumes:
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High-Throughput RNAi Screening
Methods and Protocols
Editors
David O. Azorsa
Institute of Molecular Medicine, Phoenix Children’s Hospital, Phoenix, Arizona, USA
Shilpi Arora
Constellation Pharmaceuticals, Cambridge, Massachusetts, USA
Editors
David O. Azorsa Shilpi A rora
Institute of Molecular Medicine Constellation Pharmaceuticals
Phoenix Children’s Hospital Cambridge, Massachusetts
Phoenix, Arizona USA
USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6335-5 ISBN 978-1-4939-6337-9 (eBook)
DOI 10.1007/978-1-4939-6337-9
Library of Congress Control Number: 2016945160
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Prefa ce
Ever since the discovery of small interfering RNA-mediated transcriptional inhibition by
Craig Mello and Andrew Fire, there have been great advances in manipulating this cellular
mechanism to achieve targeted gene silencing. Short interfering RNA (siRNA) and short
hairpin RNA (shRNA) are commonly used for gene silencing and validation of gene func-
tion. Both can be implemented using standard techniques of transferring nucleic acids into
cells. With the completion of the human genome project and other advances in genomics,
nucleic acid sequences that target specifi c gene transcripts have become available. In addi-
tion, both commercial and academic institutions have made advances in chemical synthesis
of double-stranded RNA and the development of large shRNA libraries. Through the use
of these libraries and high-throughput methodologies, it has become possible to advance
RNAi research from observing the phenotypic/cellular response after silencing a few genes
to silencing hundreds or thousands of genes in a single experiment. This type of high-
throughput RNAi (HT-RNAi) or loss-of-function screening has become a powerful tool in
the fi eld of functional genomics.
The power of HT-RNAi screening is in its ability to provide large amounts of functional
data for a particular biological assay. These large data sets can provide valuable insight into
the role of specifi c genes or groups of genes in a biological process. Many research groups
working with various cell models and organisms from different species have recognized the
potential of HT-RNAi assays. Studies have been done in whole organisms like C. elegans
and d rosophila as well as plant and mammalian cell systems, including established cell lines
and primary cells. HT-RNAi screening in mammalian cells has routinely been applied to
identify genes that modulate specifi c biological responses or result in a particular pheno-
type. Although there are many advantages in using HT-RNAi, there are also some potential
downsides, particularly in mammalian cell HT-RNAi screening. These include the cost of
large genome-wide libraries, the infrastructure and equipment needed to perform the
assays, and the analysis and management of large data sets.
In this volume, we cover the various aspects and methodologies for performing
HT-RNAi screens, including the use of various RNAi platforms and delivery methods in
mammalian cells, as well as various applications such as drug sensitizer identifi cation. We
also cover methods for HT-RNAi screening in non-mammalian, whole organism and cell
models including C. elegans, drosophila , S . cerevisiae, S. frugiperda, P. patens and zebrafi sh.
Finally, we examined the latest advancements in the fi elds of assay development, library
screening, data analysis, and hit selection. The goal of this volume is to provide a compre-
hensive source of protocols and other necessary information to make robust and successful
assays possible for all who wish to use HT-RNAi in their research.
Phoenix, AZ David O . Azorsa
Cambridge, MA Shilpi Arora
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v ii
1 Establishing an Infrastructure for High-Throughput
Short-I nterfering RNA Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Hongwei Yin , Chris Sereduk , and Nanyun Tang
2 Optimization of Transfection Conditions for siRNA Screening . . . . . . . . . . . . 1 5
Justin J. Montoya and David O . A zorsa
3 H igh Throughput siRNA Screening Using Reverse Transfection. . . . . . . . . . . 2 5
Carina von Schantz and Jani S aarela
4 G enome-Wide siRNA Screening Using Forward Transfection:
Identification of Modulators of Membrane Trafficking
in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9
Mariana G . Bexiga and J eremy C. S impson
5 P ooled shRNA Screening in Mammalian Cells as a Functional
Genomic Discovery Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Katarzyna J astrzebski , B astiaan E vers , and Roderick L . B eijersbergen
6 A Protocol for a High-Throughput Multiplex Cell Viability Assay. . . . . . . . . . 7 5
Daniel F. Gilbert and M ichael B outros
7 R NAi Screening of Leukemia Cells Using Electroporation. . . . . . . . . . . . . . . . 8 5
Anupriya Agarwal and Jeffrey W . T yner
8 S elf-Assembled Cell Microarray (SAMcell) for High-T hroughput
RNAi Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5
Hanshuo Z hang and Juan L i
9 I n Vitro-Pooled shRNA Screening to Identify Determinants
of Radiosensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Alessandro C eroni , Geoff S . H iggins , and Daniel V. Ebner
10 Three-Dimensional Spheroid Cell Culture Model for Target
Identification Utilizing High-Throughput RNAi Screens . . . . . . . . . . . . . . . . 121
LaKesla R . Iles and Geoffrey A . B artholomeusz
11 I n Vitro High-Throughput RNAi Screening to Accelerate
the Process of Target Identification and Drug Development . . . . . . . . . . . . . . 1 37
Hongwei Yin and M ichelle Kassner
12 H igh-Throughput, Liquid-Based Genome-Wide RNAi Screening
in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 51
Linda P . O’Reilly , Ryan R . K noerdel , G ary A. S ilverman ,
and Stephen C . Pak
13 Design and Methods of Large-Scale RNA Interference Screens
in Drosophila. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Jia Z hou and C hao T ong
vii
viii Contents
14 Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing
Lifespan and Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 71
Amit S inha and R obbie R ae
15 R NAi-Assisted Genome Evolution (RAGE) in Saccharomyces cerevisiae . . . . . . 1 83
Tong S i and H uimin Z hao
16 R NAi Screening in Spodoptera frugiperda . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 99
Subhanita Ghosh , Gatikrushna Singh , B indiya S achdev , Ajit Kumar ,
Pawan Malhotra , S unil K. M ukherjee , and Raj K. B hatnagar
17 M orphology and Gene Expression Screening with Morpholinos
in Zebrafish Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Li-Chuan T seng , Chih-Hao T ang , and Yun-Jin Jiang
18 L ive Cell Microscopy-Based RNAi Screening in the Moss
Physcomitrella patens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Tomohiro Miki , Yuki N akaoka , and Gohta G oshima
19 Data Analysis for High-Throughput RNAi Screening . . . . . . . . . . . . . . . . . . . 2 47
David O. A zorsa , M egan A . T urnidge , and Shilpi A rora
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 61
Contributors
ANUPRIYA AGARWAL • Division of Hematology and Medical Oncology,
Department of Molecular and Medical Genetics, Oregon Health and Science
University , Portland, OR , U SA ; Knight Cancer Institute, Oregon Health and
Science University , Portland, OR , USA
SHILPI ARORA • Constellation Pharmaceuticals , Lexington, MA , USA
DAVID O . AZORSA • Institute of Molecular Medicine, Phoenix Children’s Hospital ,
Phoenix, AZ, USA ; Department of Child Health, University of Arizona College of
Medicine—Phoenix , Phoenix, AZ, USA
GEOFFREY A. BARTHOLOMEUSZ • Division of Cancer Medicine, Department of
Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center ,
Houston, TX , U SA
RODERICK L. BEIJERSBERGEN • Division of Molecular Carcinogenesis and NKI Robotics
and Screening Center , The Netherlands Cancer Institute , Amsterdam,
The Netherlands
MARIANA G . BEXIGA • School of Biology and Environmental Science & UCD Conway
Institute of Biomolecular and Biomedical Research , University College Dublin ,
Dublin, Ireland
RAJ K. BHATNAGAR • Insect Resistance Group, International Centre
for Genetic Engineering and Biotechnology , New Delhi, India
MICHAEL BOUTROS • Division of Signaling and Functional Genomics, German Cancer
Research Center (DKFZ) , Heidelberg, G ermany ; Department of Cell and Molecular
Biology, Medical Faculty Mannheim , Heidelberg University , Heidelberg, Germany
ALESSANDRO CERONI • Adaptimmune Ltd , Oxford , UK
DANIEL V. EBNER • Target Discovery Institute, Nuffi eld Department of Medicine,
University of Oxford , Oxford , UK
BASTIAAN EVERS • Division of Molecular Carcinogenesis and NKI Robotics
and Screening Center , T he Netherlands Cancer Institute , Amsterdam,
The Netherlands
SUBHANITA GHOSH • Insect Resistance Group, International Centre for Genetic
Engineering and Biotechnology , N ew Delhi, India
DANIEL F. GILBERT • Institute of Medical Biotechnology and Erlangen Graduate School
in Advanced Optical Technologies (SAOT) , Friedrich-Alexander Universität
Erlangen-Nürnberg , Erlangen, Germany ; Division of Signaling and Functional
Genomics, German Cancer Research Center (DKFZ) , Heidelberg, Germany ;
Department of Cell and Molecular Biology, Medical Faculty Mannheim,
Heidelberg University , Heidelberg, Germany
GOHTA G OSHIMA • Division of Biological Science, Graduate School of Science,
Nagoya University , Nagoya, Japan
ix