Preface
RNA molecules play diverse roles in the cell owing to their secondary structure and
interaction with various proteins and nucleic acids. To study the composition, localization,
dynamics, and function of these functional RNA complexes in vitro or in live cells and tissues
of animals and plants requires the specific tagging of the RNA or of RNA-binding proteins
with suitable reporter molecules. This book provides a compendium of state-of-the-art
methods for the labeling, detection, and purification of RNA and RNA–protein complexes
and thereby constitutes an important toolbox for researchers interested in understanding
the complex roles of RNA molecules in development, signaling, and disease. In addition to
studying the natural function of RNA molecules within cells, numerous labs have developed
methods to actually apply RNA molecules as guides for the sequence-specific cleavage,
modification, or imaging of RNA and DNA. Therefore, this book also includes protocols
that apply RNA molecules as sequence-specific guide molecules.
The protocol chapters of this book are organized in six parts. Part I provides protocols
for the in situ detection of RNA molecules using fluorescent in situ hybridization (FISH)
techniques, whereas Part II provides protocols for the tagging of RNA molecules for specific
detection in live cells by fluorescent RNA-binding proteins. A dedicated review article
describes the imaging of RNA molecules with fluorogens that bind to RNA targets tagged
with specific light-up RNA aptamers. Part III contains protocols for monitoring RNA
uptake by cells or for addressing RNA transport between cells. Important protocols useful
for the characterization of RNA-binding proteins and protein complexes are presented in
Part IV. Part V is dedicated to protocols for the application of RNA molecules as guides in
RNA-mediated editing and imaging of chromosome loci. Starting with an overview article
about CRISPR guide RNA design for genome editing, this part continues by presenting
protocols in which RNA molecules are applied to guide nuclease-deactivated CRISPRassociated protein 9 (dCas9) for the imaging of specific genome loci. Another interesting
protocol in this part of the book allows the in vivo monitoring of transcribed loci by imaging
tagged nascent messenger RNA molecules emerging during gene transcription. The final
Part VI provides advanced protocols for the functional analysis of RNA molecules. These
include the detection of small RNAs involved in RNA silencing by Northern blot hybridization, the monitoring of miRNA-mediated RNA silencing events with an in vivo reporter
system, the isolation and characterization of RNA molecules associated with polyribosomes,
a protocol for transfection of RNA molecules into plant protoplasts, and also the biochemical in vitro modification and tagging of RNA molecules for imaging and structural analysis.
The experimental protocols are provided by leading experts with hands-on experience in
the respective method. I hope that the provision of these protocols will further stimulate
research in RNA biology and that the research community interested in this field will accept
this book as an important reference.
I am very grateful to all the authors who contributed to this book. I also thank the series
editor, John M. Walker, for his continuous support in developing this volume.
Strasbourg, France
Manfred Heinlein
v
RNA molecules play diverse roles in the cell owing to their secondary structure and
interaction with various proteins and nucleic acids. To study the composition, localization,
dynamics, and function of these functional RNA complexes in vitro or in live cells and tissues
of animals and plants requires the specific tagging of the RNA or of RNA-binding proteins
with suitable reporter molecules. This book provides a compendium of state-of-the-art
methods for the labeling, detection, and purification of RNA and RNA–protein complexes
and thereby constitutes an important toolbox for researchers interested in understanding
the complex roles of RNA molecules in development, signaling, and disease. In addition to
studying the natural function of RNA molecules within cells, numerous labs have developed
methods to actually apply RNA molecules as guides for the sequence-specific cleavage,
modification, or imaging of RNA and DNA. Therefore, this book also includes protocols
that apply RNA molecules as sequence-specific guide molecules.
The protocol chapters of this book are organized in six parts. Part I provides protocols
for the in situ detection of RNA molecules using fluorescent in situ hybridization (FISH)
techniques, whereas Part II provides protocols for the tagging of RNA molecules for specific
detection in live cells by fluorescent RNA-binding proteins. A dedicated review article
describes the imaging of RNA molecules with fluorogens that bind to RNA targets tagged
with specific light-up RNA aptamers. Part III contains protocols for monitoring RNA
uptake by cells or for addressing RNA transport between cells. Important protocols useful
for the characterization of RNA-binding proteins and protein complexes are presented in
Part IV. Part V is dedicated to protocols for the application of RNA molecules as guides in
RNA-mediated editing and imaging of chromosome loci. Starting with an overview article
about CRISPR guide RNA design for genome editing, this part continues by presenting
protocols in which RNA molecules are applied to guide nuclease-deactivated CRISPRassociated protein 9 (dCas9) for the imaging of specific genome loci. Another interesting
protocol in this part of the book allows the in vivo monitoring of transcribed loci by imaging
tagged nascent messenger RNA molecules emerging during gene transcription. The final
Part VI provides advanced protocols for the functional analysis of RNA molecules. These
include the detection of small RNAs involved in RNA silencing by Northern blot hybridization, the monitoring of miRNA-mediated RNA silencing events with an in vivo reporter
system, the isolation and characterization of RNA molecules associated with polyribosomes,
a protocol for transfection of RNA molecules into plant protoplasts, and also the biochemical in vitro modification and tagging of RNA molecules for imaging and structural analysis.
The experimental protocols are provided by leading experts with hands-on experience in
the respective method. I hope that the provision of these protocols will further stimulate
research in RNA biology and that the research community interested in this field will accept
this book as an important reference.
I am very grateful to all the authors who contributed to this book. I also thank the series
editor, John M. Walker, for his continuous support in developing this volume.
Strasbourg, France
Manfred Heinlein
v