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Methods in
Molecular Biology 2300

Mathieu Rederstorff Editor

Small Non-
Coding RNAs
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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Small Non-Coding RNAs

Methods and Protocols

Second Edition

Edited by

Mathieu Rederstorff
Université de Lorraine, CNRS, IMoPA, Nancy, France
Editor
Mathieu Rederstorff
Université de Lorraine
CNRS, IMoPA
Nancy, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1385-6 ISBN 978-1-0716-1386-3 (eBook)
https://doi.org/10.1007/978-1-0716-1386-3

© Springer Science+Business Media, LLC, part of Springer Nature 2015, 2021

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Preface

Small Non-Coding RNAs: Methods and Protocols is a laboratory protocols book dedicated to
biochemists or cellular/molecular biologists, already working in the field of RNA biology or
willing to start studying small non-coding RNAs in their projects.
Owing to their small sizes, tri-dimensional structures, low abundances, or differential
expression levels, small non-coding RNAs require customized dedicated protocols for their
identification and study, compared, for instance, to messenger RNAs (mRNAs).
This volume contains basic as well as more sophisticated, state-of-the-art methods to
tackle all aspects of small non-coding RNA biology, from their isolation and precipitation,
through their quantification, accurate detection, functional analysis, to their high-
throughput or even structural analysis.
This survey of technologies will be of valuable help to all the researchers interested in
studying the numerous functions of small non-coding RNAs.
I sincerely thank all the authors for their contribution to this exciting second edition.

Nancy, France Mathieu Rederstorff

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I INTRODUCTION TO SMALL NON-CODING RNAS

1 Regulatory Non-Coding RNAs: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Camille Virciglio, Yoann Abel, and Mathieu Rederstorff
2 RNA Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Valérie Bourguignon-Igel, Yoann Abel, and Mathieu Rederstorff
3 Quantitative and Qualitative Assessment of Small RNA Preparations . . . . . . . . . . 17
Virginie Marchand, Lilia Ayadi, Valérie Bourguignon-Igel,
and Yuri Motorin
4 Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA
GapmeRs Transfection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Charbel Alfeghaly, Christelle Aigueperse, Sylvain Maenner,
and Isabelle Behm-Ansmant

PART II DETECTION OF SMALL NON-CODING RNAS

5 Northern Blot Detection of Tiny RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Jana C. Wiegard, M. Amri C. Schlüter, Olga Y. Burenina,
Elena A. Kubareva, Gabriele Klug, Arnold Grünweller,
and Roland K. Hartmann
6 Stem-Loop qRT-PCR–Based Quantification of miRNAs . . . . . . . . . . . . . . . . . . . . . 59
Yoann Abel and Mathieu Rederstorff
7 Detection and Labeling of Small Non-Coding RNAs by Splinted Ligation . . . . . 65
Marlène Vayssières, Gabrielle Bourgeois, Florian Chardon, Anne-Sophie
Tillault, and Magali Blaud
8 Fluorescence In Situ Hybridization of Small Non-Coding RNAs . . . . . . . . . . . . . 73
Valentin Vautrot, Géraud Heckler, Christelle Aigueperse,
and Isabelle Behm-Ansmant

PART III FUNCTIONAL ANALYSIS OF SMALL NON-CODING RNAS

9 Using Native RIP, UV-CLIP or fCLIP to Address Protein–RNA
Interactions In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Laeya Baldini and Stéphane Labialle
10 Purification of RiboNucleoProtein Particles by MS2-MBP Affinity
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Aymeric Sanchez and Sylvain Maenner

vii
viii Contents

11 Study of Genome-Wide Occupancy of Long Non-Coding RNAs

Using Chromatin Isolation by RNA Purification (ChIRP) . . . . . . . . . . . . . . . . . . . 107
Charbel Alfeghaly, Isabelle Behm-Ansmant, and Sylvain Maenner
12 Gene Reporter Assays to Study miRNA Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Yoann Abel and Mathieu Rederstorff
13 CRISPR/Cas9 System to Knockdown MicroRNA In Vitro
and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Bin Yi, Kristina Larter, and Yaguang Xi

PART IV HIGH-THROUGHPUT ANALYSIS OF SMALL NON-CODING RNAS

14 Microarray Analysis of Whole-Transcriptome RNAs Including

Non-Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Quentin Thuillier and Isabelle Behm-Ansmant
15 Isolation, Extraction and Deep-Sequencing Analysis of Extracellular
RNAs (exRNAs) from Human Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Virginie Marchand, Adeline Galvanin, and Yuri Motorin
16 Analysis of the RNA and Protein Complexome by Grad-seq . . . . . . . . . . . . . . . . . 183
Jens Hör and Jörg Vogel
17 μIVC-Seq: A Method for Ultrahigh-Throughput Development
and Functional Characterization of Small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Farah Bouhedda, Roger Cubi, Stéphanie Baudrey,
and Michael Ryckelynck

PART V STRUCTURAL APPROACHES

18 RNA Secondary Structure Study by Chemical Probing Methods

Using DMS and CMCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Fatima Alghoul, Gilbert Eriani, and Franck Martin
19 Application of NMR Spectroscopy to Determine the 3D Structure
of Small Non-Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Marie-Eve Chagot, Marc Quinternet, Xavier Manival,
and Isabelle Lebars

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Contributors

YOANN ABEL • IGMM, CNRS, Université de Montpellier, Montpellier, France

CHRISTELLE AIGUEPERSE • Université de Lorraine, CNRS, IMoPA, Nancy, France
CHARBEL ALFEGHALY • Université de Lorraine, CNRS, IMoPA, Nancy, France
FATIMA ALGHOUL • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
LILIA AYADI • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France;
Université de Lorraine, CNRS, IMoPA, Nancy, France
LAEYA BALDINI • Université de Lorraine, CNRS, IMoPA, Nancy, France
STÉPHANIE BAUDREY • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France
ISABELLE BEHM-ANSMANT • Université de Lorraine, CNRS, IMoPA, Nancy, France
MAGALI BLAUD • Laboratoire CiTCoM “Cibles Thérapeutique et Conception des Mé
dicaments”, CNRS UMR 8038, Faculté de Pharmacie, Université de Paris, Paris, France
FARAH BOUHEDDA • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France
GABRIELLE BOURGEOIS • Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique,
Palaiseau, France
VALÉRIE BOURGUIGNON-IGEL • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy,
France; Université de Lorraine, CNRS, IMoPA, Nancy, France
OLGA Y. BURENINA • Center of Life Sciences, Skolkovo Institute of Science and Technology,
Moscow, Russia; Chemistry Department and A.N. Belozersky Institute of Physico-Chemical
Biology, M.V. Lomonosov Moscow State University, Moscow, Russia
MARIE-EVE CHAGOT • Université de Lorraine, CNRS, IMoPA, Nancy, France
FLORIAN CHARDON • ZMBE Zentrum für Molekularbuiologie der Entzündung, Münster,
Germany
ROGER CUBI • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR
9002, Strasbourg, France
GILBERT ERIANI • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
ADELINE GALVANIN • Université de Lorraine, CNRS, IMoPA, Nancy, France
ARNOLD GRÜNWELLER • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
ROLAND K. HARTMANN • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
GÉRAUD HECKLER • Université de Lorraine, CNRS, IMoPA, Nancy, France
JENS HÖR • Institute of Molecular Infection Biology, University of Würzburg, Würzburg,
Germany
GABRIELE KLUG • Institute of Microbiology and Molecular Biology, Justus-Liebig-University-
Gießen, Gießen, Germany
ELENA A. KUBAREVA • Chemistry Department and A.N. Belozersky Institute of Physico-
Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia
STÉPHANE LABIALLE • Université de Lorraine, CNRS, IMoPA, Nancy, France

ix
x Contributors

KRISTINA LARTER • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana
State University Health Sciences Center, New Orleans, LA, USA
ISABELLE LEBARS • Université de Strasbourg, CNRS, ARN UPR 9002, 2 allée Conrad
Roentgen, Strasbourg, France
SYLVAIN MAENNER • Université de Lorraine, CNRS, IMoPA, Nancy, France
XAVIER MANIVAL • Université de Lorraine, CNRS, IMoPA, Nancy, France
VIRGINIE MARCHAND • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France
FRANCK MARTIN • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
YURI MOTORIN • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France;
Université de Lorraine, CNRS, IMoPA, Nancy, France
MARC QUINTERNET • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France
MATHIEU REDERSTORFF • Université de Lorraine, CNRS, IMoPA, Nancy, France
MICHAEL RYCKELYNCK • Université de Strasbourg, CNRS, Architecture et Réactivité de
l’ARN, UPR 9002, Strasbourg, France
AYMERIC SANCHEZ • Université de Lorraine, CNRS, IMoPA, Nancy, France
M. AMRI C. SCHLÜTER • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
QUENTIN THUILLIER • Université de Lorraine, CNRS, IMoPA, Nancy, France
ANNE-SOPHIE TILLAULT • Department of Chemistry and Biochemistry, Alberta RNA
Research and Training Institute, University of Lethbridge, Lethbridge, AB, Canada
VALENTIN VAUTROT • Université de Lorraine, CNRS, IMoPA, Nancy, France
MARLÈNE VAYSSIÈRES • Laboratoire CiTCoM “Cibles Thérapeutique et Conception des Mé
dicaments”, CNRS UMR 8038, Faculté de Pharmacie, Université de Paris, Paris, France
CAMILLE VIRCIGLIO • Université de Lorraine, CNRS, IMoPA, Nancy, France
JÖRG VOGEL • Institute of Molecular Infection Biology, University of Würzburg, Würzburg,
Germany; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz
Centre for Infection Research (HZI), Würzburg, Germany
JANA C. WIEGARD • Institute of Pharmaceutical Chemistry, Philipps-University Marburg,
Marburg, Germany
YAGUANG XI • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State
University Health Sciences Center, New Orleans, LA, USA
BIN YI • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State
University Health Sciences Center, New Orleans, LA, USA
 Part I

Introduction to Small Non-Coding RNAs

 Chapter 1

Regulatory Non-Coding RNAs: An Overview

Camille Virciglio, Yoann Abel, and Mathieu Rederstorff

Abstract
The discovery of new classes of non-coding RNAs has always been preceded or accompanied by techno-
logical breakthroughs, and these outstanding progresses in transcriptomics approaches enabled to regularly
add new members to the list. From the first detection of tRNAs, through the revolution of miRNAs
discovery, to the recent identification of eRNAs or the identification of new functions for already known
ncRNAs, this introductive review provides a very concise historical and functional overview of most
prominent small regulatory non-coding RNA families.

Key words miRNA, snoRNA, piRNA, tRNA, rRNA, tiRNA, snRN, eRNA, ceRNA, circRNA

1 Small Non-Coding RNAs Begin

It has been more than 60 years that the first soluble cytoplasmic
small non-coding RNA (sncRNA), a yeast alanine transfer RNA
(tRNA), was identified and its primary structure determined
[1, 2]. Together with ribosomal RNAs (rRNA), the nucleic acids
moieties of the ribosomes, tRNAs play a central role in protein
synthesis, as the adaptor molecules providing the correct amino
acid in response to a specific codon on the mRNA [3]. Only
20 years later were the first nuclear RNAs uncovered: uridine-rich
RNAs, or U-RNAs, were observed to be very abundant, nucleus, or
sometimes even nucleolus specific and highly conserved [4, 5];
small nuclear RNAs (snRNA) U1, U2, U4, U5, and U6 were
later shown to be involved in splicing, in association with several
proteins within a ribonucleoprotein particle (RNP) called spliceo-
some [6]. Base pairings among snRNAs and between snRNAs and
pre-messenger RNA (pre-mRNA) regions account for the proper
conformation of both spliceosome and pre-mRNA, eventually lead-
ing to release of the introns and mature mRNAs [7]. Interestingly,
most small nucleolar RNAs (snoRNAs) are processed from the
released introns [8]. Within snoRNPs, snoRNAs guide post-
transcriptional RNA modifications of other ncRNAs, e.g., rRNAs

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

3
4 Camille Virciglio et al.

and snRNAs: 20 -O-methylations for C/D boxes snoRNAs and

pseudouridylations for H/ACA boxes snoRNAs. Some snoRNAs,
such as U3 or U8, are instead involved in early cleavages of
pre-rRNA during ribosome biogenesis in the nucleolus [9–11].
In the late 1990s, pioneering studies aimed at identifying novel
non-coding RNAs using experimental approaches combining
library generation and sequencing identified numerous novel snoR-
NAs in various models including human, using material that was
usually discarded: small total RNA in the size range of 50 to
500 nucleotides [12]. At that time, looking for shorter RNAs was
considered a nonsense, and they were therefore disregarded. Non-
sense? Antisense actually!

2 miRNAs in the Spotlight

In the early 1990s, the lin-14 gene was shown to be involved in

temporal development in the worm C. elegans. The lin-4 gene
product, most probably a polypeptide, was shown to negatively
regulate lin-14 expression by directly acting on the 30 untranslated
region of its pre-mRNA [13]. In 1993, both Ambros and Ruvkun
labs discovered that the lin-4 gene product actually was a small
non-coding RNA, of 22 nucleotides in length in its mature form,
targeting the 30 UTR of the lin-14 pre-mRNA several times, owing
to imperfect antisense base pairing [14, 15]. Unfortunately
enough, apparently, the lin-4 small RNA neither had additional
counterparts in worm nor was it found to be conserved in other
species. However, in 2000, Pasquinelli and coworkers identified
that let-7, another 22 nucleotides long small temporal RNA
(stRNA) involved in C. elegans developmental timing, was con-
served in human and drosophila, anticipating the likely discovery
of many other small regulatory RNAs [16]. Revolution came less
than 1 year later, when Tuschl, Bartel, and Ambros labs reported, in
the same issue of Science, the observation of an abundant class of
novel small regulatory RNA in worm, human, and drosophila,
termed since microRNAs (miRNAs) [17–19]. Even certain viruses
were next shown to encode their own miRNAs [20, 21].
It was rapidly observed that the miRNA maturation and assem-
bly machinery was the same as the one implicated in the formation
of short interfering RNAs (siRNAs), involved in the RNA interfer-
ence (RNAi) pathway described in worm a couple of years earlier by
the 2006 Nobel Prize awardees Andrew Fire and Craig Mello
[22, 23], and even earlier in plants [24]. miRNA/siRNA matura-
tion, from a double-stranded precursor, is now well understood
[25], but still open to surprises. It was, for example, discovered that
miRNAs could be processed from other types of ncRNAs, such as
snoRNAs [11, 26, 27], with possible novel functions still to be
deciphered.
 ncRNAs Overview 5

In human, siRNAs, within the RNA-induced silencing complex

(RISC), mediate perfectly complementary mRNA target cleavages
[28, 29]. On the other hand, miRNAs regulate gene expression by
imperfectly base-pairing to the 30 UTR of the targeted mRNA,
repressing its translation, which eventually leads to the mRNA
decay by different possible mechanisms [30, 31].
However, another degree of complexity has emerged recently,
as miRNA-dependent gene expression regulation was shown to be
modulated by so-called RNA “sponges” [32], also referred to as
competitive endogenous RNAs (ceRNAs) [33, 34]. Many ceRNAs
compete with miRNA-binding sites and microRNA recognition
elements (MREs). They sequester miRNAs, which creates networks
of shared miRNAs and enables modulation of miRNA-dependent
gene expression regulation [33].
The family of circular RNAs, or circRNAs, already observed in
the early 1990s [35, 36], recently regained more interest as possible
ceRNAs members [37–39]. Their formation is generally consecu-
tive to the alternative splicing of a pre-mRNA, in a process called
“back-splicing.” In this process, the upstream splice acceptor is
connected to a downstream splice donor [40, 41]. Other functions
have been proposed for circRNAs, one of which leading to the idea
that circRNAs might also act as RNA-binding proteins “sponges”
[42] or even compete with the mRNA splicing itself [43].

3 Small Non-Coding RNAs Return

Another sncRNA family, the Piwi interacting RNA (piRNAs), dis-

covered about half a decade after miRNAs, also regulate transcrip-
tion [44]. piRNAs are 27–29 nucleotides long RNAs. They prevent
transposons dissemination in germinal cell lines owing to an origi-
nal “ping-pong” mechanism [45, 46]. The smallest ncRNAs
known to date, the transcription initiation RNAs (tiRNAs), sized
17–18 nucleotides, are generated upon stalling or backtracking of
RNA polymerase II (RNAPII) near the transcription start sites
(TSSs), and might be involved in transcription initiation regulation
as well [47, 48]. TSSs appear to be the source of bunches of small
and long non-coding RNAs (lncRNAs) [49], such as transcription
start site-associated RNAs (TSSa-RNAs) and promoter-associated
RNAs (PASRs) in animals [50], or promoter upstream transcripts
(PROMPTs) [51] and cryptic/Xrn1-sensitive unstable transcripts
(CUTs/XUTs) in yeast [52–54]. While lncRNAs are generally
involved in epigenetic and chromatin modifications [55–57], func-
tionality of short transcripts is debated. They might be involved in
maintaining chromatin into a transcriptionally active state or in
maintaining RNAPII available near TSSs [58].
6 Camille Virciglio et al.

In this respect, the recently described enhancer RNAs (eRNAs)

may feature some common functions with TSS-associated RNAs.
Enhancer elements are generally short DNA sequences promoting
transcription and were initially considered not to be transcribed
themselves, until recently [59, 60]. eRNAs’ biogenesis was since
shown to be initiated by the recruitment of activators and transcrip-
tion factors normally required for the transcription of surrounding
genes. Next, histone reorganization at the enhancer site is activated
by epigenetic modifications, which increase accessibility of the
enhancer sequence, triggering initiation of transcription, elonga-
tion, and processing of eRNAs [61]. In terms of functions, it was
demonstrated that eRNAs altered chromatin architecture and epi-
genetic modifications and could recruit proteins to the enhancers
regions [61]. In addition, transcription of eRNAs, rather than
eRNAs themselves, was shown to attenuate and fine-tune transcrip-
tion of surrounding mRNA by interfering with or pausing the
transcription machinery [62, 63].

Acknowledgments

This work was supported by the Centre National pour la Recherche

Scientifique, the Université de Lorraine and the Région Lorraine.

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 Chapter 2

RNA Precipitation
Valérie Bourguignon-Igel, Yoann Abel, and Mathieu Rederstorff

Abstract
Precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of
alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous
variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt,
alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA
recovery.

Key words RNA, Precipitation, Alcohol, Salt, Carrier

1 Introduction

Alcoholic precipitation is used to recover, purify, or concentrate

nucleic acids from a total extract. It relies on nucleic acids differen-
tial solubility in solution upon addition of alcohol and salts. Nucleic
acids are soluble in water because both nucleic acids and water are
polar molecules that can therefore interact together [1]. The nega-
tively charged phosphate groups (PO3 ) of nucleic acids electro-
statically interact with water. Hence, water molecules form a
solvation or hydration shell around each nucleic acid molecule,
isolating them from each other and subsequently dissolving them
in solution. Alcohol and salt enable to disrupt this hydration shell.
In water, salts used for nucleic acids precipitation completely disso-
ciate into their constituting anions and cations. Cations neutralize
the negative charges of nucleic acids (PO3 groups) while alcohol,
which has a much lower dielectric constant than water, increases the
electrostatic interaction force between the phosphate groups and
the cations. Once neutralized, nucleic acids become less hydrophilic
and thus precipitate out of solution. Additionally, alcohol decreases
the repulsive forces between the inter-helical phosphates so that
nucleic acids molecules aggregate.
Depending on the sample type or the subsequent experiments
to be performed, several parameters such as the type of salt or

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

11
12 Valérie Bourguignon-Igel et al.

alcohol to be used, the addition of a carrier, the precipitation

temperature, or the centrifugation speed have to be considered to
improve RNA quality and recovery yield (see Notes 1–5).

2 Materials

1. 100% ethanol.
2. 70% ethanol.
3. 100% isopropanol.
4. 3 M sodium acetate, pH 5.2.
5. 5 M ammonium acetate.
6. 8 M lithium chloride.
7. 2 M sodium chloride.
8. Yeast tRNA (10–20 μg/ml).
9. Salmon sperm DNA (10–20 μg/ml).
10. Glycogen (50–150 μg/ml).
11. GlycoBlue™ Co-precipitant (50–150 μg/ml).
12. Linear polyacrylamide (10–20 μg/ml).
13. 1 M MgCl2.
14. RNase-free, DEPC-treated water.
15. 1  TE buffer, pH 8.0: 10 mM Tris–HCl, pH 8.0,
1 mM EDTA.

3 Methods

To avoid RNase contaminations when preparing and handling

RNAs, always use appropriate precautions such as RNase-free
water (see Note 6), gloves, RNase-free tubes and tips.

3.1 Ethanol 1. Adjust sample volume to a minimum of 100 μl (see Note 7).
Precipitation 2. Add sodium acetate to the sample to a final concentration of
0.3 M (about 1/10 of volume) (see Notes 1, 3 and 8).
3. Add 2.5 to 3 volumes of ice-cold 100% ethanol (see Note 2).
4. Mix thoroughly by inverting the tube or pipetting up and
down (see Note 9).
5. Incubate on ice for 15 min to 1 h (see Note 4).
6. Centrifuge at 12,000  g for 30 min at 4  C (see Note 5).
7. Carefully discard the supernatant without disturbing the pellet.
8. Wash the RNA pellet with 0.5 ml of 70% ethanol and mix
gently.
 RNA Precipitation 13

9. Centrifuge at 12,000  g for 15 min at 4  C.

10. Carefully discard the supernatant without disturbing the pellet
(see Note 10).
11. Air-dry the RNA pellet for 5–10 min at room temperature (see
Note 11).
12. Dissolve the pellet in RNase-free water (see Note 12).

4 Notes

1. Different salts can be used during alcoholic precipitation, the

choice of which depending on the subsequent applications.
Indeed, each salt features specific properties providing different
advantages and/or drawbacks [1, 2]. Sodium acetate (0.3 M
final concentration, pH 5.2) is the most widely used salt for
alcoholic precipitation [1, 2]. However, if the sample contains
SDS, sodium chloride (0.2 M final concentration) could be
alternatively chosen. In this case, SDS would remain soluble
in ethanol [1, 2], which would allow its separation and removal
from the RNA. Ammonium acetate (2.5 M final concentration)
is another possible choice. It allows to greatly reduce
co-precipitation of both dNTPs and oligosaccharides. How-
ever, ammonium acetate should not be used if a phosphoryla-
tion reaction is planned after precipitation since ammonium
ions inhibit T4 polynucleotide kinase [1, 2]. Finally, a last
broadly used salt for RNA precipitation is lithium chloride
(0.8 M final concentration). It is very soluble in alcohol and
therefore co-precipitates with RNA to a smaller extent than
other salts [1, 2]. Moreover, DNA, proteins, and carbohydrates
do not efficiently precipitate with lithium chloride, which
therefore leads to a greater purity of RNAs precipitated with
this salt [3]. Unfortunately, possible loss of small RNAs have
been reported (100 nt or less, e.g., miRNAs, tRNAs, sn- and
snoRNAs or 5S/5.8S rRNAs). Anyway, as small and large
RNAs feature different solubility in high ionic strength solu-
tions, small RNAs remain soluble while larger ones do not. This
property enables to selectively purify small RNAs with high
concentrations of LiCl (up to 2.5 M) even without alcohol
[1], as small RNAs will remain in solution. However, lithium
chloride should not be employed if subsequent reverse tran-
scription or in vitro translation reactions are planned, as the
chloride ions at this concentration inhibit RNA-dependent
DNA polymerase as well as initiation of protein synthesis in
most cell-free systems [1].
2. Alternatively, isopropanol can replace ethanol as a precipitation
alcohol. Indeed, since isopropanol is less polar than ethanol,
14 Valérie Bourguignon-Igel et al.

RNAs are even less soluble in isopropanol than they are in

ethanol. Therefore, for a similar precipitation efficiency, a
lower volume of isopropanol is necessary [1, 2]. Isopropanol
can therefore replace ethanol in case of large sample volume,
using about one volume of isopropanol per volume of sample.
However, as salts are also less soluble in isopropanol, they
coprecipitate and contaminate RNA preparation to a higher
extent [2]. Therefore, washing steps after precipitation with
isopropanol are very important. Finally, isopropanol is less
volatile than ethanol and will require more time for the RNA
pellet to dry [2].
3. If you expect RNA quantity to be very low or if RNAs are very
small in size (<100 nucleotides), you can add a carrier or
co-precipitant to increase recovery yield before incubation on
ice. Also, if you do not see any pellet after the centrifugation
step, it is possible to proceed again to precipitation (starting
from step 5, Subheading 3.1) after addition of a carrier. Car-
riers are molecules that trigger precipitation of RNA as they are
insoluble in alcohol and thus precipitate. After centrifugation,
the pellet will consequently be bigger and thus more visible,
which will greatly facilitate removal of the supernatant without
affecting the pellet. Moreover, colored dyes can be covalently
linked to carriers, which additionally facilitates the observation
of the pellet.
The choice of the carrier in alcoholic precipitation is impor-
tant. As for the choice of the salt to be used, carrier choice will
depend on the subsequent reactions to be performed [2]. The
most frequently used carrier molecules are yeast tRNAs (final
concentration of 10–20 μg/ml), glycogen (final concentration
of 50–150 μg/ml), or linear polyacrylamide (final concentra-
tion of 10–20 μg/ml). Yeast tRNAs are biologically active
RNAs that might interfere with some subsequent current
molecular biology reactions. For example, tailing reactions or
reactions catalyzed by polynucleotide kinases or terminal trans-
ferases are inhibited by large amounts of yeast tRNAs [4]. In
some cases, template-dependant cDNA synthesis is inhibited as
well [5]. On the other hand, glycogen is an inert, DNA/RNA-
free molecule. It interferes neither with subsequent enzymatic
reactions (PCR, digestion, ligation, reverse transcription, label-
ing, transcription, hybridization, etc.) nor with electrophoresis
or spectrophotometrical measurements [6]. Glycogen even
allows improved precipitation of very small RNA (>8 nt).
However, glycogen may prevent reverse transcription in the
case of very large templates, as well as nucleic acids interactions
with proteins [7] in a concentration-dependent manner
[8]. Finally, linear polyacrylamide is another DNA/RNA-free
inert molecule. As glycogen, it does not interfere neither with
 RNA Precipitation 15

enzymatic reactions such as phosphorylation by polynucleotide

kinase, ligation, cDNA synthesis, in vitro transcription, diges-
tion by endonucleases nor with electrophoresis [9]. Linear
polyacrylamide does not inhibit nucleic acids interactions
with proteins [10]; however, it does not trigger
co-precipitation of very small RNAs (20 bases) [7].
4. The incubation step for the alcoholic precipitation of RNAs is
generally performed at very low temperature ( 20  C
or 80  C) in most protocols. However, although possible
RNases might be less active at lower temperatures, this appears
to be rather counter-productive [1, 2]. Indeed, the lower the
temperature, the higher the viscosity, which decreases RNAs
movements and thus aggregation and precipitation. Addition-
ally, the dielectric constant increases when the temperature
diminishes, therefore precipitation efficiency decreases with
the temperature. Finally, salts solubility decreases with the
temperature as well, which leads to stronger coprecipitation
of salts at lower temperatures [11]. Therefore, incubation at
room temperature or on ice is sufficient and recommended,
providing you work in RNase-free conditions. If you retrieve a
low amount of RNA or if you do not see the pellet, you can
increase the incubation time or add a carrier if not done already.
An average time of about 15 min is generally sufficient.
5. If the amount of RNA is low, if you do not see the pellet, or if
you work with very small RNAs (<100 nucleotides), you can
both increase the speed and time of centrifugation in order to
allow the pellet to more firmly stick to the tube wall [1, 2].
6. Water can be treated with DEPC (diethylpyrocarbonate) to
avoid RNases contaminations.
7. Too small volumes of samples reduce the yield of RNA
recovery.
8. MgCl2 should be added to a final concentration of 10 mM for
small RNAs (<100 nucleotides) [1].
9. Do not vortex. Too vigorous mixing could damage and slice
the RNA.
10. Great care must be taken when discarding the supernatant in
order not to lose the RNA pellet.
11. Do not dry the pellet too much, especially employing a speed-
vacuum, as too dry RNA pellets are more difficult to
resolubilize.
12. You can dissolve the pellet in 1  TE buffer, pH 8, instead of
water. Although EDTA chelates divalent cations, thus inhibit-
ing metalloenzymes such as nucleases or proteases, it might
also inhibit polymerases or other enzymes necessary for your
subsequent experiences.
16 Valérie Bourguignon-Igel et al.
References
1. Sambrook J, Fritsch EF, Maniatis T (1989)
Molecular cloning, a laboratory manual, 2nd
edn. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, pp E.10–E.15
2. Zumbo P (2012) Ethanol precipitation.
Department of Physiology & Biophysics,
Weill Cornell Medical College
3. Barlow J et al (2001) A simple method for the
quantitative isolation of Undegraded high
molecular weight ribonucleic acid. Biochem
Biophys Res Commun 13:61–66
4. Michelson AM, Orkin SH (1982) Characteri-
zation of the Homopolymer tailing reaction
catalyzed by terminal deoxynucleotidyl trans-
ferase. J Biol Chem 257:14773–14782
5. Wang QT et al (2002) Yeast tRNA as carrier in
the isolation of microscale RNA for global
amplification and expression profiling. Bio-
Techniques 33:788–796
6. Tracy S (1981) Improved rapid methodology
for the isolation oh nucleic acids from agarose
gels. Prep Biochem 11:251–268
7. Gaillard C, Strauss F (1990) Ethanol precipita-
tion of DNA with linear polyacrylamide as car-
rier. Nucleic Acids Res 18:378
8. Baugh LR et al (2001) Quantitative analysis of
mRNA amplification by in vitro transcription.
Nucleic Acids Res 29:E29
9. Aruffo A, Seed B (1987) Molecular cloning of
a CD28 cDNA by a high-efficiency COS cell
expression system. Proc Natl Acad Sci
84:8573–8577
10. Strauss F, Varshavsky A (1984) A protein binds
to a satellite DNA repeat at three specific sites
that would be brought into mutual proximity
by DNA folding in the nucleosome. Cell
37:889–901
11. Zeugin J, Hartley JL (1985) Ethanol precipita-
tion of DNA. Focus 7:1–2
 Chapter 3

Quantitative and Qualitative Assessment of Small RNA

Preparations
Virginie Marchand, Lilia Ayadi, Valérie Bourguignon-Igel,
and Yuri Motorin

Abstract
Recent advances in high-throughput sequencing have shed new light on the diversity of small noncoding
RNA (sncRNA) classes and their crucial roles in gene regulation and disease. One key step in sncRNA
profiling consists in their quantification and assessment of their degradation extent. In this chapter, we will
describe different gold standard methods used to achieve both purposes before using the sncRNAs in
downstream applications.

Key words RNA quantification, UV spectroscopy, Fluorescence, Capillary electrophoresis, RNA

integrity number

1 Introduction

The small noncoding RNA (sncRNAs) family is composed of dif-

ferent RNA subclasses including tRNAs and tRNA-derived frag-
ments (tRFs), miRNAs, piRNAs, snRNAS, snoRNAs, and
scaRNAs. High-throughput sequencing methods have revealed
extreme diversity of sncRNAs, many of which being initially con-
sidered as simple degradation products. Hence, RNA quantifica-
tion is an important and necessary step prior to any further analysis.
The most commonly used technique for measuring the concentra-
tion of nucleic acid samples is the determination of their absorbance
at 260 nm (A260). While this is a very fast and cheap method to
estimate RNA quantity and its relative purity, this technique reveals
to be rather inaccurate if the sncRNA sample is contaminated by
DNA, proteins, or other products such as rNTPs, carbohydrates,
phenol, DTT, or chaotropic salts, which compromise accuracy of
the optical density readings. To avoid these problems, other meth-
ods, such as fluorometry, have been developed. These methods
employ specific fluorescent dyes to selectively quantify a

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021

17
18 Virginie Marchand et al.

biomolecule of interest (RNA, DNA, or proteins). As these dyes

only emit signals when bound to their specific target molecule, even
at low concentrations, this enables, for instance, to distinguish
between RNA and DNA present in the same sample. Use of fluo-
rescence for RNA quantification is very robust, easy, and rapid,
moreover it tolerates various buffer compositions, even detergent-
containing buffers (see Note 1). The contamination of sncRNA
samples by proteins and/or DNA can also be measured using
protein- and/or DNA-specific kits. However, contamination by
other substances, such as phenol, cannot be detected this way.
Next, RNA quality can be assessed by two additional
approaches: conventional denaturing/non-denaturing agarose gel
electrophoresis or different versions of high-resolution capillary
electrophoresis.
Since most sncRNAs are only present in low amounts in total
RNA preparations, their integrity is difficult to evaluate directly. In
addition, length of sncRNAs may considerably vary. Thus, evalua-
tion of sncRNA, as well as mRNAs, quality in total RNA samples is
deduced from global integrity of abundant RNA species, mostly
rRNAs and, possibly, tRNAs.
Rapid assessment of RNA quality can easily be obtained by
running a sample on denaturing agarose gel [1] and inspecting
the 28S/18S rRNA ratio, but this technique requires tremendous
amount of input RNA and does not provide quantitative results. In
addition, the use of toxic and/or carcinogenic denaturing agents
such as formaldehyde, formamide, or urea has led to the develop-
ment of alternative methods, such as native agarose gels supple-
mented with sodium hypochlorite, sometimes referred to as bleach
agarose gels [2] (see Note 2).
To overcome these limitations, microfluidic-based technolo-
gies have been developed to quantify and analyze RNA integrity
in an unbiased way. Instruments such as the Agilent 2100 Bioana-
lyzer, 2200 TapeStation (Agilent Technologies), or the Experion
(Bio-Rad) are considered the gold standards for measuring RNA
quality at the picogram scale. The 28S/18S ratio is automatically
calculated and, when comprised between 1.8 and 2.0, indicates
good RNA integrity. In addition, an integrated algorithm assigns
an RNA Integrity Number (RIN, Agilent Technologies) from 1 to
10, where 10 represents a completely intact RNA and 1 a highly
degraded one [3, 4]. However, running of microfluidic chips is
relatively expensive, and some technical limitations exist as well.
Calculation of RIN is meaningful only for total RNA samples,
containing major peaks of rRNA. If the RNA preparation does
not contain rRNAs, RIN is erroneous or not calculated at all,
making it impossible to conclude on the quality of the RNA prepa-
ration. Another problem concerns RNA preparations of insect or
plant origin, as the profile for total RNA differs substantially from
other common eukaryotic sources. In plant leaves, total RNA
 Quantity and Quality Control of Small RNAs 19

contains a high proportion of rRNA species from chloroplasts (16S,

23S, and 5S rRNAs) [5], while in insects, e.g., Drosophila, the 28S
rRNA is processed in two pieces that migrate around the 18S upon
heat denaturation [6] (Fig. 2a).
This chapter focuses on the most common methods to quantify
and estimate the quality of an RNA preparation and provides several
tips to optimize these methods in order to get accurate and repro-
ducible results.

2 Materials

Prepare all solutions using RNase-free water. Wear gloves to pre-

vent degradation of RNA samples by RNases.

2.1 RNA 1. UV-visible small volume spectrophotometer: Any kind of

Quantification UV-visible spectrophotometer allowing measurements of 1 μl
samples (e.g., NanoDrop One (see Note 3) or One-C, Nano-
2.1.1 Using
Drop 2000, 2000c, or 8000, Thermo Fischer Scientific). We
a Spectrophotometer
use a NanoDrop One.
2. 1.5 ml RNase-free microcentrifuge tubes.
3. RNase-free water.

2.1.2 Using 1. Any kind of fluorometer able to quantify RNA, DNA, and
a Fluorometer proteins with high sensitivity, e.g., Qubit 3.0 Fluorometer
(Life technologies) which is the one we use.
2. Thin-walled polypropylene tubes of 500 μl, compatible with
the fluorometer (e.g., Qubit Assay Tube or Axygen PCR-05-C
tubes, VWR)
3. RNA assay kit: Qubit RNA Assay Kit (5–100 ng), Qubit RNA
BR Assay Kit (20–1000 ng), Qubit microRNA Assay Kit
(1–500 ng).
4. Optional: Qubit dsDNA HS Assay Kit (0.2–100 ng) and Qubit
Protein Assay Kit (0.25–5 μg).

2.2 RNA Quality 1. Agarose.

Assessment 2. 10 TBE buffer: 108 mg/ml Tris–HCl, pH 8, 55 mg/ml
2.2.1 Native Agarose Gel boric acid, 9.3 mg/ml EDTA, pH 8.
Electrophoresis 3. Agarose gel electrophoresis chamber.
4. Ethidium bromide (EtBr) or equivalent (e.g., GelRed, FluoP-
robes, or SYBR Safe DNA Gel Stain, Invitrogen).
5. 10x DNA loading buffer: 1.9 mM xylene cyanol, 1.5 mM
bromophenol blue, 25% glycerol.
20 Virginie Marchand et al.

2.2.2 Capillary 1. Agilent 2100 Bioanalyzer (Agilent Technologies) or Experion

Electrophoresis (Bio-Rad). Since we use an Agilent 2100 Bioanalyzer, this
chapter will focus on this machine.
2. Agilent RNA 6000 Nano Kit (25 to 500 ng/μl), Agilent RNA
6000 Pico kit (50 to 5000 pg/μl), or Agilent Small RNA kit
(50 to 2000 pg/μl) (Agilent) (Table 1).
3. Chip priming station (Agilent Technologies).
4. Heating block.
5. RNase-free water.
6. RNase-free 1.5 ml microcentrifuge tubes.

3 Methods

3.1 RNA Carry out all procedures at room temperature unless otherwise
Quantification specified.

3.1.1 Using 1. Select the “Nucleic Acids” tab from the home screen of the
a Spectrophotometer NanoDrop One spectrophotometer.
2. Pipette and load 1 μl of a blank solution (e.g., RNase-free water
or TE buffer) onto the lower pedestal and lower the arm.
3. If “Auto-blank” is On, the blank measurement starts automat-
ically, otherwise select “Blank” and wait for the measurement
to complete.
4. Lift the arm and clean both pedestals with a clean wipe.
5. Pipette and load 1 μl of the sample solution onto the pedestal,
lower the arm (see Note 4).
6. If “Auto-Measure” is On, the sample measurement starts auto-
matically, otherwise select “Measure” and wait for the measure-
ment to complete. The spectrum and reported values are
displayed.
7. When you are done with sample measurements, select “End
Experiment” and export data on a USB stick.
8. Lift the arm and clean both pedestals with a new wipe.
9. Analyze the data obtained for your different RNA samples. The
concentration of your RNA sample based on the absorbance at
260 nm is given in “ng/μl” by default setting. The “A260/
A280” ratio is provided. This ratio is used to roughly assess the
purity of your RNA preparation. A ratio of 2 is generally
considered as “pure” RNA. The “A260/A230” ratio is
provided as well. This ratio is used to assess RNA purity. For
“pure” RNAs, it should be in the range of 1.8–2.2 (see Note 5).
In addition, if you use the NanoDrop One, its technology
will enable to identify most contaminants (phenol, proteins,
etc.).
 Quantity and Quality Control of Small RNAs 21

Table 1
Overview of the technical specifications for the Agilent RNA 6000 Nano, RNA 6000 Pico, and Small
RNA kits

Agilent RNA 6000 Pico Agilent Small RNA

Agilent RNA 6000 Nano kit kit kit
Reagents and consumables included in the kit (stable for 4 months)
Number of chips/kit 25 chips
Electrode cleaner 3 chips
chips
RNA dye concentrate 1 vial (blue cap)
RNA marker 4 vials (green cap)
RNA conditioning N.A 1 vial (white cap)
solution
Gel matrix 2 vials (red cap)
Spin filters 4 spin filters 2 spin filters
Ladder 1 vial (yellow cap)
Safe-lock Eppendorf 30
PCR tubes
Syringe 1
Technical specifications
Samples analyzed/ 12 11
chip
Analysis range (nt) 20–6000 nt 6–150 nt
Sample volume 1
Quantitative range 25–500 ng/μL (5–500 ng/μ 0.2–5 ng/μl (0.05–5 ng/ 0.05–2 ng/μl for
(qualitative range) L) for total RNA μl) for total RNA purified miRNA
25–250 ng/μL (25–250 ng/μ 0.5–5 ng/μl (0.25–5 ng/
L) for mRNA μl) for mRNA
Reproducibility of 10% CV 20% CV
quantitation
Quantitation accuracy 20% CV (for ladder as sample) 30% CV (for ladder as 25% CV (for ladder
sample) as sample)
Buffer compatibility 100 mM Tris or 125 mM 50 mM Tris or 50 mM 10 mM Tris and
NaCl or 15 mM MgCl2 NaCl 0.1 mM EDTA
Analysis run time 30
(min)
22 Virginie Marchand et al.

3.1.2 Using 1. Equilibrate all solutions of the appropriate kit at room temper-
a Fluorometer ature for at least 30 min before starting the experiments. The
kit provides the concentrated assay reagent, dilution buffer,
and pre-diluted standards (see Note 6).
2. Prepare the dye working solution by diluting the concentrated
assay reagent 200 times in dilution buffer. Prepare 200 μl of
working solution for each sample and the two standards.
3. Prepare the two standards annotated “C” and “D” by mixing
10 μl of standard with 190 μl of working solution.
4. Add working solution up to 200 μl to 1–20 μl of RNA sample.
5. Vortex the tubes for 2 s and incubate them for 2 min at room
temperature.
6. On the home screen of the Qubit 3.0 Fluorometer, select the
quantification assay “RNA” for which you want to perform a
new calibration.
7. Select “Read standards” to perform a calibration.
8. When indicated, insert the standard #1 tube and select “Read
standard.” Standard #1 and #2 correspond to standards “C”
and “D,” respectively.
9. Once the calibration is done, select “Run samples” to make the
measurements.
10. Indicate with the + and buttons on the wheel, the sample
volume added to the assay tube and select the units for the
output sample concentration.
11. Insert a sample tube into the sample chamber, close the lid and
then select “Read tube.”
12. Read the results, remove the current sample and insert a
new one.
13. To address if your RNA sample preparation is contaminated by
DNA or proteins, repeat the experiment with the DNA and/or
Protein kits (see Note 7).

3.2 RNA Quality 1. Pour a 1% w/v agarose gel in 1 TBE.

Assessment 2. Heat at least 1 μg of each RNA samples for 2 min at 70  C and
3.2.1 Native Agarose Gel cool down on ice.
Electrophoresis 3. Mix with 10 DNA loading buffer.
4. Load an RNA ladder or an RNA control on the gel to estimate
RNA quantity and quality.
5. Run the gel at 10 V/cm in 1 TBE running buffer.
6. Stain the gel in a 1 GelRed or SYBR Safe DNA Gel stain bath
for 15 min.
7. Using a UV transilluminator, analyze the quality of the RNAs
on the gel.
 Quantity and Quality Control of Small RNAs 23

3.2.2 Capillary 1. Prepare the ladder provided in the kit: Spin down the tube and
Electrophoresis transfer 10 μl of it to a RNase-free tube. Heat for 2 min at
70  C. Cool down on ice and add 90 μl of RNase-free water.
Prepare 5 μl aliquots using the Safe-Lock PCR Tubes provided
in the kit and store them at 70  C. Before use, thaw one tube
and keep it on ice (Fig. 1a) (see Note 8).
2. Equilibrate all solutions of the kit at room temperature for at
least 30 min before starting the experiments (see Note 6).
3. Place 550 μl (Agilent RNA 6000 Nano or Pico kit) or 650 μl
(Agilent Small RNA kit) of the gel matrix (red cap vial) into a
dedicated spin filter. Spin at room temperature for 10 min at
1500  g (Agilent RNA 6000 Nano or Pico kit) or for 15 min
at 10,000  g (Agilent Small RNA kit) (Table 1) (Fig. 1b).
4. Prepare 65 μl (Agilent RNA 6000 Nano or Pico kit) or 40 μl
(Agilent Small RNA kit) aliquots of the gel and store them at
4  C for a maximum of 1 month.
5. To avoid contamination of your RNA samples by RNases, clean
the electrodes before and after each experiment by filling one of
the wells of the electrode cleaner with 350 μl of fresh RNase-
free water (see Note 9).
6. Place the electrode cleaner in the Agilent 2100 bioanalyzer and
wait for 5 min before removing it.
7. Wait for 30 min to allow evaporation of the water from the
electrodes (see Note 10).
8. Prepare the gel–dye mix by adding 1 μl (Agilent RNA 6000
Nano or Pico kit) or 2 μl (Agilent Small RNA kit) of RNA dye
concentrate (blue cap vial) to a gel aliquot. Mix by pipetting up
and down and centrifuge for 10 min at 13,000  g at room
temperature (Fig. 1c).
9. During centrifugation, prepare your samples as follows: Dilute
your RNA samples with RNase-free water to be within the
optimal range concentration of the assay (Table 1).
10. Heat the diluted RNA samples for 2 min at 70  C to prevent
formation of secondary structures and cool them down on ice
(Fig. 1d).
11. Add 1 μl of your diluted RNA samples to 11 (or 12) tubes of
1.5 ml containing 5 μl of RNA marker (green cap vial) (see
Notes 11 and 12) (Table 1). Mix by pipetting up and down
(Fig. 1d).
12. Mix 1 μl of the ladder with 5 μl of RNA marker (green cap vial)
in a 1.5 ml tube (see Note 13) (Table 1). Mix by pipetting up
and down (Fig. 1d).
24 Virginie Marchand et al.

a) e)

1-3 G

4-6 G

7-9 G

Heat for 2 min Cool down 10-11 CS

at 70°C on ice

RNA Pico Chip

10 μl Add 90 μl of H2O Aliquot by 5 μl

ladder (RNase-free) (store at -70°C)

Load 9 μl of Gel-dye mix

G
b) G

CS

Close chip priming station

Centrifuge for Press the plunger of the syringe
Load on
10 min at 13,000 g at RT until it is hold by the clip
Wait for 30s
Release the clip and
550 μl Aliquot by 65 μl wait for the plunger to go up
gel matrix (store at 4°C) Open the chip priming station

Load 9 μl of Gel-dye mix

c) G

CS

+ 65 μl gel
aliquot
Centrifuge for
Load 9 μl of Conditioning Solution
10 min at 13,000 g at RT
G

G
1 μl Gel-dye mix ready
dye concentrate G

CS

d) Load 6 μl of Diluted Ladder

G

G
5 μl ladder G
aliquot Mix by pipetting
CS
up and down
6 μl diluted ladder

1 μl ladder
Load 6 μl of Diluted RNA sample
G

CS
+ + +
12 empty
1.5 ml tubes

Inspect for air bubbles or

liquid spilling in the wells

Heat for 2 min Mix by pipetting Place the chip in the

x μl at 70°C 1 μl up and down 6 μl diluted sample Agilent 2100 Bioanalyzer
(0.5-5 ng/μl) and start the run

Fig. 1 Quick guide for RNA Pico Chip preparation and loading. (a) Ladder preparation. (b) Gel preparation. (c)
Gel–dye mix preparation. (d) Samples and ladder preparation. (e) RNA Pico Chip loading
 Quantity and Quality Control of Small RNAs 25

13. Prepare the chip priming station before loading the gel–dye
mix. Check that the base plate is adjusted in its default position
(C position) (Fig. 1e).
14. Open a new syringe, slide it into the hole of the lock adapter
and screw it to the priming station. Adjust the plunger to 1 ml
(Fig. 1e).
15. Adjust the syringe clip to the highest top (Agilent RNA 6000
Nano or Pico kit) or to the lowest top (Agilent Small RNA kit)
position (see Table 1).
16. Once you are ready, load 9 μl of the gel–dye mix in the well
marked with a “G” surrounded by a black circle (see Note 14)
(Fig. 1e).
17. Close properly the chip priming station (see Note 15) and press
the plunger of the syringe until it is hold by the clip (Fig. 1e).
18. Wait exactly for 30 s (Agilent RNA 6000 Nano or Pico kit) or
60 s (Agilent Small RNA kit) and then release the clip.
19. Wait for 5 s until the plunger stops its upward move and pull it
slowly back to the 1 ml position.
20. Open the chip priming station and load 9 μl of gel–dye mix in
the other wells marked with a “G.”
21. Load 9 μl of the conditioning solution (not for the Agilent
RNA 6000 Nano kit) in the well marked “CS” (Fig. 1e).
22. Load 6 μl of the diluted ladder in the well marked with a ladder
(Fig. 1e).
23. Load 6 μl of the diluted RNA samples in the wells marked 1–11
(for Agilent RNA 6000 Pico and Small RNA kit) or 1–12 (for
Agilent RNA 6000 Nano kit) (Fig. 1e).
24. Inspect the chip and make sure that no liquid is present on the
edges of the wells. If any, pipet it away (see Note 14).
25. Insert the chip in the Agilent 2100 Bioanalyzer.
26. Carefully close the lid. The electrodes of the cartridge will enter
into the wells of the chip (see Note 15).
27. The 2100 expert software screen shows that you have inserted
a chip by displaying a chip icon on the top left corner of the
screen.
28. Select the appropriate assay you want to perform (e.g., eukary-
otic total RNA Pico series II).
29. Press “Start” to begin the chip run (see Note 16).
30. After the run, immediately remove the chip and clean the
electrodes (see Note 10).
31. Analyze the results of the chip (Fig. 2).
26 Virginie Marchand et al.

a A
RN tal R
NA
total to
t n
ec uma
ins h
Total RNA extracted from human PBMC cells (RIN=8.7)
[FU]
200

100

0
(nt)
25 500 4000 [nt] 4000
Total RNA extracted from insect cells (RIN=NA)
[FU] 2000
200 1000
500
200
0
25
25 500 4000 [nt]
RIN NA 8.7
b
Total RNA extracted from human PBMC cells (RIN=9.3)
[FU]
18S rRNA 28S rRNA
re
NA xtu
200 lR mi NA
ota NA m iR
100
sRNAs nt tt R
ed
ma a s rifi
hu ye pu
0
25 500 4000 [nt]
yeast tRNA mixture (RIN=2.6)
[FU]

1000
(nt)
500
4000
0
25 500 4000 [nt] 2000
Synthetic miRNA (RIN=2.6)
1000
[FU]
500
200

25
0
RIN 9.3 2.6 2.6
25 500 4000 [nt]

c [FU] Total RNA extracted from human PBMC cells (RIN=9.3)

50 miRNA tRNA
re
NA xtu
lR mi NA
ota NA m iR
0 nt t tR ed
ma as rifi
hu ye pu
4 20 60 100 150 [nt]

[FU] yeast tRNA mixture (RIN=2.6)

(nt)
0
4 20 60 100 150 [nt] 150

[FU] Synthetic miRNA (RIN=2.6)

100
200 80
60
100
40
0 20

4 20 60 100 150 [nt] 4

miRNA tRNA

Fig. 2 Examples of RNA profiles obtained using the Agilent 2100 Bioanalyzer. (a) High-quality insect or human
total RNA obtained after Trizol extraction analyzed on an RNA Pico Chip. The human total RNA trace profile is
 Quantity and Quality Control of Small RNAs 27

4 Notes

1. Several possible contaminants were tested in the concentration

range of 25–500 ng/ml. The corresponding data are provided
in the manufacturer’s protocol. For instance, presence of up to
0.01% SDS, 1% ethanol, 0.001% Triton X-100, 5 mM NaCl, or
1 mM MgCl2 in the RNA sample preparation is tolerated and
does not affect the accuracy of the quantification.
2. In both cases, if RNA degradation is observed, it is difficult to
assess whether this is due to poor quality of the RNA prepara-
tion or because RNases are present on the gel electrophoresis
device and degraded the sample while running. Incorporating
chlorine bleach (6% sodium hypochlorite) into a standard TAE
or TBE agarose gel helps preventing the latter problem [2].
3. The NanoDrop One is equipped with the “Acclaro Sample
Intelligence” technology. This feature helps to identify the
type of contaminant present in a sample, determine the level
of contamination, and obtain a corrected nucleic acid
concentration.
4. Make sure the sample is well centered on the bottom pedestal
and avoid air bubbles. Loading of 1.2 μl of RNA could be an
alternative to get more accurate and reproducible results.
5. Using RNase-free water instead of 10 mM Tris-EDTA (TE),
pH 8.0 to dilute your sample may lower the 260/280 ratio
below 2.0 due to the lower pH of water [7]. A 260/280 ratio
of 1.8 for samples diluted in RNase-free water is considered
“pure” for RNA.
6. The concentrated assay reagent contains DMSO, which is a
potential mutagen, therefore wear hand and eye protection and

Fig. 2 (continued) typical of a high-quality RNA since the 28S and 18S rRNA peaks are present and dominant.
A RIN (RNA Integrity Number) of 8.7 was attributed to the RNA sample, which is indicative of good quality. Note
the presence of abundant small RNAs including 5.8S and 5S rRNAs, tRNAs, and other noncoding RNAs ranging
from 25 to 200 nts. The insect total RNA trace profile is also typical of a high-quality RNA sample, even though
it looked like degraded and no RIN could be attributed since the 28S rRNA peak is absent. Indeed, upon heat
denaturation, the insect 28S rRNA is cleaved leading to fragments migrating with the 18S rRNA. (b) RNA trace
profiles obtained on a Pico RNA chip for a yeast total tRNA preparation and a synthetic miRNAs sample (21 nt)
were compared to a human total RNA preparation. As expected, yeast tRNA mixture and synthetic miRNA both
migrate as the small RNAs of the human total RNA preparation. (c) RNA trace profiles obtained on a Small RNA
chip for a yeast total tRNA preparation and a synthetic miRNAs sample (21 nt) were compared to a human total
RNA preparation. The human total RNA preparation leads to three major peaks, one between 50 and 80 nts,
corresponding mainly to tRNAs, one around 90 nts corresponding to snoRNAs, and one around 140 nts
corresponding to the 5.8S rRNA. 5S rRNA corresponds to a small pic at 120 nts. Though miRNAs are detected
in this sample, they represent too small a portion of total RNAs and are therefore not visible on the RNA trace
profile
28 Virginie Marchand et al.

be careful when preparing and handling reagents and samples.

Store this reagent at room temperature, desiccated and pro-
tected from light. Store the dilution buffer at room tempera-
ture and the standards at 4  C. All kit components are stable for
6 months.
7. In the protein kit, there are 3 standards and samples should be
incubated for 15 min instead of 2 min with kits dedicated to
nucleic acids.
8. We recommend doing this immediately upon kit arrival to
reduce the number of freeze and thaw events.
9. RNase contamination problems of the electrodes are quite
frequent and will affect the RIN of your sample. Therefore, if
the Agilent 2100 Bioanalyzer is also used for DNA assays, it is
strongly recommended to use a dedicated electrode cartridge
for RNA assays. In addition, we recommend for each chip to
load an internal RNA control (total RNA preparation with a
known RIN > 9). If you encounter contamination problems,
clean the electrode cartridge with an RNA Zap solution for at
least 10 min, then rinse the electrodes with RNase-free water
and let them dry out for at least one night.
10. Water on the electrodes will lead to abnormal RNA profiles.
Make sure any traces of water are evaporated before running
the chip.
11. To avoid RNase contamination problems, we recommend
labeling the RNA marker vial currently in use, and to split it
into aliquots. If you have any doubt concerning a possible
RNase contamination, trash the current vial and use a new
aliquot.
12. Do not leave any empty well otherwise the chip will not run
properly. If you do not have enough samples to load, use
RNase-free water instead and proceed as usual.
13. The ladder is quite stable at 70  C and may be used beyond
4 months.
14. The gel and samples loading steps on the chip are crucial. Insert
the tip of the pipette at the very bottom of the well when
loading gel or samples. This will prevent formation of air
bubbles. Placing the pipette at the wall or the edge of the
well will lead to poor results.
15. The chip priming station is properly closed only when you hear
a clear “click” sound.
16. The Agilent 2100 Bioanalyzer is very sensitive to vibrations,
therefore install it on a dedicated bench and make sure that no
vibrations will occur during the run.
 Quantity and Quality Control of Small RNAs
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2. Aranda PS, LaJoie DM, Jorcyk CL (2012)
Bleach gel: a simple agarose gel for analyzing
RNA quality. Electrophoresis 33:366–369
3. Becker C, Hammerle-Fickinger A, Riedmaier I,
Pfaffl MW (2010) mRNA and microRNA qual-
ity control for RT-qPCR analysis. Methods
50:237–243
4. Schroeder A, Mueller O, Stocker S, Salowsky R,
Leiber M, Gassmann M, Lightfoot S, Menzel W,
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Granzow M, Ragg T (2006) The RIN: an RNA
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 Chapter 4

Non-Coding RNA Silencing in Mammalian Cells by Antisense

LNA GapmeRs Transfection
Charbel Alfeghaly, Christelle Aigueperse, Sylvain Maenner,
and Isabelle Behm-Ansmant

Abstract
The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies.
However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not
efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to
RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless
of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL
and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection
methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Key words lncRNA silencing, Antisense LNA GapmeRs, Calcium phosphate transfection, Lipofecta-
mineTM 2000, ANRIL, SATIII

1 Introduction

Despite recent advances in our understanding of noncoding RNAs’

roles, most of them and more especially long noncoding RNAs
(lncRNAs) functions remain poorly understood. Loss-of-function
analyses are essential in deciphering the functions of ncRNAs by
assessing the biological consequences of suppressing their expres-
sion. However, many ncRNAs are confined to the nucleus and thus
are inefficiently targeted by the cytoplasmic RNA-induced silencing
complex (RISC) [1, 2]. To overcome this limitation, antisense LNA
GapmeRs were designed to provide an effective knockdown of both
coding and noncoding RNAs with a size greater than 80 nucleo-
tides, regardless of their cellular localization. Antisense LNA Gap-
meRs are single-stranded antisense oligonucleotides (ASOs) that
catalyze nuclear RNase H-dependent degradation of complemen-
tary RNA targets (Fig. 1). They are 16 nucleotides long and are
enriched with LNA (Locked Nucleic Acids) nucleotides in the
flanking regions and DNA nucleotides in an LNA-free central

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021

31
32 Charbel Alfeghaly et al.

Transcription

ncRNA
LNA DNA LNA
Antisense LNA GapmeR
Nucleus
RNaseH
RNase
RNase H cleavage

Degraded ncRNA

Fig. 1 Silencing of nuclear noncoding RNA using antisense LNA GapmeRs.

Antisense LNA GapmeRs are single-stranded oligonucleotides containing a
DNA portion flanked by LNA. They hybridize by complementarity to their RNA
targets. The DNA:RNA duplexes are recognized by nuclear RNase H, creating an
endonucleolytic cleavage in the RNA strand. The two generated fragments are
degraded by exonucleases. The antisense LNA GapmeR is then released and will
guide degradation of another RNA strand. (Adapted from (https://www.qiagen.
com))

gap. LNA are a class of high-affinity RNA analogs in which the

ribose ring is “locked” in the ideal conformation for Watson–Crick
binding thanks to a 2-O,40 -C-methylene bridge [3]. As a result,
LNA-containing oligonucleotides exhibit higher thermal stability
(Tm) when hybridized to a complementary DNA or RNA strand,
regardless of the GC contents. Moreover, incorporation of LNA
bases suppresses innate immune stimulation [4] and confers resis-
tance to endonucleases and exonucleases, which leads to higher
in vitro and in vivo stability. This resistance to enzymatic degrada-
tion is further increased by the fact that antisense LNA GapmeRs
have fully modified phosphorothioate (PS) backbones. Moreover,
antisense LNA GapmeRs work independently of the RISC complex
and thus have no miRNA-like off-target effects. As they are single
stranded, they guarantee strand-specific knockdown, which is of
particular interest when studying ncRNAs that derive from tran-
scriptionally complex loci with overlapping sense and antisense
transcripts.
 Non-Coding RNA Silencing 33

Due to their small size and stability in culture media, antisense

LNA GapmeRs can be taken up by cells without the need for a
transfection agent. Nevertheless, non-assisted uptake does require
higher concentrations of antisense LNA GapmeR than would be
needed when using transfection agents, and the knockdown kinet-
ics would be slower. In this chapter, we describe the two transfec-
tion protocols commonly used in our lab to efficiently transfect
antisense LNA GapmeRs. First, calcium phosphate-mediated trans-
fection, a biochemical method, is a simple handling and inexpensive
procedure. Calcium phosphate forms an insoluble coprecipitate
with DNA, which is absorbed by endocytosis or phagocytosis by
the cells [5]. This technique has proven to be very effective for the
delivery of oligonucleotides (siRNA, ASOs) in our hands. Another
delivery system is LipofectamineTM 2000, a liposomal cationic lipid
reagent, which forms a complex with the DNA. The resulting stable
cationic complexes adhere to the negatively charged cell membrane
before fusing with them [6]. LipofectamineTM 2000 is known to be
a highly efficient carrier of LNA oligonucleotides to cells nuclei [7].
In this chapter, we illustrate these efficient transfection meth-
ods by silencing two nuclear lncRNAs: SATIII and ANRIL. SATIII
lncRNAs are transcribed from the pericentromeric SATIII repeats
located on human chromosomes 9, 12, 15 [8]. Rosic et al. [9]
showed that Drosophila melanogaster SATIII RNAs, on the X chro-
mosome, constitute an integral part of centromere identity and are
essential for kinetochore formation and cell division. In addition,
SATIII lncRNAs promote intron retention during thermal stress
recovery [10]. On the other hand, the ANRIL lncRNA is tran-
scribed from the 9p21 locus and is alternatively spliced into several
isoforms harboring different combination of exons. ANRIL was
shown to promote in cis the epigenetic silencing of the CDKN2A
and CDKN2B tumor suppressor genes and hence to promote cell
proliferation [11, 12]. Using calcium phosphate-mediated trans-
fection, we efficiently silenced the expression of human SATIII and
ANRIL lncRNAs (Fig. 2a, b). Rapid effects were observed for
SATIII silencing as extinction appeared as soon as 6 h after treat-
ment with antisense LNA GapmeRs. On the contrary, 48 h were
needed to reach an efficient ANRIL silencing using two consecutive
treatments with antisense LNA GapmeRs independently of the
transfection method used (Fig. 2b, c).
To conclude, antisense LNA GapmeRs containing a central
DNA segment flanked by LNA gaps offer an alternative to
siRNA-dependent technology for suppression of nuclear noncod-
ing RNA expression, with the calcium phosphate-mediated trans-
fection being the most efficient and cheapest transfection method.
 34 Charbel Alfeghaly et al.

A. B. C.
100 LNA CTL 100 LNA CTL 100 LNA CTL
LNA SATIII 6h LNA ANRIL LNA ANRIL

Relative RNA expression (%)

Relative RNA expression (%)

Relative RNA expression (%)

LNA SATIII 24h n=3 n=1

80 80 80
n=3

60 60 60

40 40 40
19 25
19.9
20 20 20
4.8
0 0 0
SATIII ANRIL ANRIL

Calcium phosphate Lipofectamine

Fig. 2 Silencing of SATIII and ANRIL lncRNAs using specific antisense LNA GapmeRs. (a) Calcium phosphate-
mediated transfection of HeLa S3 cells is used to deliver either LNA GapmeRs specifically targeting SATIII RNA
(LNA SATIII) or LNA oligonucleotides against the GFP gene (LNA CTL) as a negative control. The efficiency of
SATIII knockdown is monitored by RT-qPCR. Transfection of SATIII LNA dramatically reduces the expression
level of SATIII RNAs and this effect is greater 6 h post-transfection compared to 24 h. Values are calculated
from three independent experiments and normalized against GAPDH mRNA level. The level of the SATIII RNA is
expressed relatively to the level of the control (100%). (b) Calcium phosphate-mediated transfection of
HEK293 cells is used to deliver either LNA GapmeRs specifically targeting ANRIL (LNA ANRIL) or a scrambled
sequence (LNA CTL) as a negative control. Efficiency of ANRIL knockdown is monitored by RT-qPCR.
Transfection of LNA ANRIL reduces ANRIL expression up to 80% 48 h post-transfection. Values are calculated
from three independent experiments and normalized against GAPDH mRNA levels. The level of ANRIL is
expressed relatively to the level of the control (100%). (c) Lipofectamine 2000 is used to transfect HEK293
cells with either LNA GapmeRs specifically targeting ANRIL (LNA ANRIL) or a scrambled sequence (LNA CTL) as
a negative control. Efficiency of ANRIL knockdown is monitored by RT-qPCR, which shows an efficient
reduction in ANRIL’s expression up to 75%, similarly to the calcium phosphate method. Values are calculated
from one experiment and normalized against GAPDH mRNA levels. The level of ANRIL is expressed relatively to
the level of the control (100%). Means  SEM are shown

2 Materials

2.1 Mammalian Cell 1. HeLa S3 cells and HEK293 cells (ATCC) (see Note 1).
Culture 2. DMEM (Dulbecco’s Modified Eagle Medium) cell culture
medium, supplemented with 10% of fetal calf serum, 2 mM
L-glutamine, 100 U/ml of penicillin, and 100 μg/ml of
streptomycin.
3. Six-well plates (for 100-mm dishes, reagent quantities are mul-
tiplied by 6).
 Non-Coding RNA Silencing 35

2.2 Antisense LNA Chemically modified antisense LNA GapmeRs are produced by
GapmeRs Exiqon-Qiagen, resuspended in nuclease-free H2O to a concentra-
(Exiqon-Qiagen) tion between 50 and 100 μM (see Note 2) and stored as aliquots at
20  C to avoid repeated freeze and thaw cycles.

2.3 Standard 1. TE (0.1, pH 7.6): 1 mM Tris–HCl, pH 8.0, 0.1 mM EDTA.

Calcium Phosphate 2. 2 M calcium chloride (CaCl2). Sterilize by filtration and store
Procedure frozen as 1 ml aliquots at 20  C.
3. 2 HEPES-buffered saline (HBS): 50 mM HEPES, 0.28 M
NaCl, 10 mM KCl, 1.5 mM Na2HPO4, pH 7.05 (see Note 3).
Sterilize by filtration and store frozen as 50 ml aliquots at
20  C.

2.4 Lipofectamine 1. Lipofectamine™ 2000 Transfection Reagent (Life

2000 Procedure Technologies).
2. Gibco™ Opti-MEM™ I Reduced Serum Medium (Life
Technologies).

3 Methods

3.1 Calcium 1. One day prior to transfection, seed 3  105 exponentially

Phosphate growing cells in 2 ml DMEM medium in 6-well plates. Incu-
Transfection bate cells for 20–24 h at 37  C with 5% CO2 (see Note 4).
2. One to 5 h before transfection, replace the medium with 2 ml
of fresh growth medium.
3. For each individual transfection in 6-well plates, dilute 3 μl of
50 μM SATIII antisense LNA GapmeRs or 1 μL of 50 μM
ANRIL antisense LNA GapmeRs with 0.1X TE to a final
volume of 36.8 μl and combine with 5.2 μl of 2 M CaCl2.
4. Add an equal volume of 2 HBS (42 μl in this case) with a
pipette.
5. Mix by pipetting up and down about 10 times to inject air
bubbles, which enhances the formation of the transfection
precipitate.
6. Incubate for 20 min at room temperature (see Note 5).
7. Add the 84 μl of transfection mixture drop by drop in each well
(see Note 6).
8. After 4–16 h of incubation, remove the DNA precipitate con-
taining medium and replace it with warm complete growth
medium (see Note 7).
9. Return the cells to the incubator for 2–96 h at 37  C with 5%
CO2 (see Note 8 and 9).
36 Charbel Alfeghaly et al.

3.2 Lipofectamine™ 1. One day prior to transfection, seed 5  105 exponentially

2000 Transfection growing cells in 2 ml of DMEM medium in 6-well plates.
Incubate cells for 20–24 h at 37  C with 5% CO2 (see Note 10).
2. One to 5 h before transfection, replace the medium with 2 ml
of fresh growth medium.
3. For each individual transfection in 6-well plates, dilute 1 μl of
50 μM antisense LNA GapmeRs with 249 μL of Gibco™ Opti-
MEM™ I Reduced Serum Medium. Mix well by pipetting.
4. In a separate tube, dilute 5 μL of Lipofectamine™ 2000
reagent in 245 μL of Gibco™ Opti-MEM™ I Reduced
Serum Medium. Mix well by pipetting.
5. Incubate for 5 min at room temperature.
6. Add the Lipofectamine mix to the solution containing the LNA
GapmeRs and mix gently by pipetting.
7. Incubate for 20 min at room temperature.
8. Add the 500 μl of transfection mixture drop by drop in
each well.
9. Return the cells to the incubator for 24–48 h at 37  C with 5%
CO2 (see Note 8).

4 Notes

1. Other cell lines can easily be transfected as well.

2. A master mix can be assembled if multiple transfections of the
same antisense LNA GapmeRs have to be performed. For
ANRIL, a mix of 4 LNA GapmeRs hybridizing to different
ANRIL regions was used at 12.5 μM each (50 μM total).
3. Reagent consistency is critical to achieve a high transfection
efficiency and reproducibility; small changes in pH can affect
transfection efficiency.
4. It is critical that the cell density ranges between 30% and 60%
confluency at the day of transfection.
5. Longer incubation times can result in the formation of fewer
but larger precipitates that reduce transfection efficiency.
6. For the depletion of SATIII and ANRIL lncRNAs, cells were
transfected with 75 nM and 25 nM of antisense LNA Gap-
meRs, respectively, into 2 ml of medium. The concentration of
antisense LNA GapmeRs to be used is usually in the 1–100 nM
range and depends on the abundance of the targeted RNA in
the cells.
7. Prolonged cell contact with calcium phosphate precipitates
may result in cytotoxicity.
 Non-Coding RNA Silencing 37

8. Antisense LNA GapmeRs should be tested to find out the best

experimental conditions to get a maximal silencing efficiency. If
the effect is too weak, you can proceed with a second transfec-
tion after 24–48 h to prolong the effect. If necessary, cells
should be split prior to the second transfection. For the deple-
tion of ANRIL, two rounds of transfection were done indepen-
dently of the transfection method used. The second round was
performed 24 h after the first one. Cells were collected 48 h
after the second transfection.
9. For the depletion of SATIII RNAs, 6 h of transfection were
sufficient.
10. It is critical that the cell density ranges between 50% and 70%
confluency on the day of transfection.

Acknowledgments

This work was supported partly by the french PIA project “Lorraine
Université d’Excellence” (ANR-15IDEX-04-LUE) and the RHU
program FIGHT-HF (ANR-15-RHU-004). The CNRS and UL
are also thanked for fundings.

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 Part II

Detection of Small Non-Coding RNAs

Another random document with
no related content on Scribd:
 The time came when Dr. Mateer had a shop equipped to do a
great variety of work; and though not on a large scale, yet big
enough to meet his needs. Already in 1886 in a letter to his brother
William he said: “In order to repair apparatus, and in order to make
many simpler articles, I have fitted up quite a complete workshop,
entirely at my own expense. I have invested in the shop, in tools and
materials quite one thousand dollars. I keep a workman at my own
cost, whom I have trained so that he can do most ordinary kinds of
work. There are a great many small articles we can make here more
cheaply than we can buy them. There are, however, many articles
we cannot make, especially those that involve glass or the use of
special machinery, or special skill.” That shop continued to grow, and
the variety of its output increased. Writing of this, Mrs. Ada Mateer
says:

So soon as possible in addition to the room used for

carpenter work, a side house was devoted to the purposes of
a shop, which grew in completeness as time went on. An
upper story was used for storing finished apparatus, for a
painting, varnishing, and drying room. The lower story was
the shop proper, with well, smithy, a long workroom, private
room for chemicals and so forth. Every conceivable amount of
space in the shop—above, around, and below—was occupied
with materials, on boards hung from above, in cases made of
old boxes lining the walls, and on the floor. The shop
contained not only materials for things that are to be, but
became also a tomb of things that were, but are not, as well
as a hospital for things disabled. What old histories were
unearthed when, after forty years, this shop had to be moved
to Wei Hsien!

Up there it was perpetuated, the main difference consisting in

larger and better quarters, with some improved conveniences. His
wife continues:
 For every machine bought, the market was canvassed by
correspondence, and the best selected. Especially was this
true with reference to any tools or machinery used in the
construction of apparatus,—as machines for turning,
blacksmithing, plumbing, screw-cutting, burnishing,
electroplating, casting, and so forth. His shop was thoroughly
fitted with all appliances for the making of apparatus, or
electric or steam outfitting, so that he was ready to do
anything, from setting up a windmill or water system, or
installing an engine and dynamo, to brazing broken spectacle
frames or repairing a bicycle.

So far as it was practicable he turned over the actual mechanical

labor to Chinese workmen,—a skilled foreman and apprentices
under the foreman’s direction.
Why, though a missionary, did he employ so considerable a part of
his time in this way? Especially at the outset of his missionary career
stern necessity to meet his own needs and those of his associates
drove him to this line of work. Had he been set down in China at
some such place as Shanghai, where foreign articles could be
purchased, very likely his mechanical gifts would have remained
largely dormant. But at Tengchow he helped to make a stove out of
odds and ends, because one was indispensable in order to keep
warm. For the same sort of reason he extracted teeth and made
false sets, cobbled shoes, and acted as master workman of all the
building trades in the erection of the “new home.” Sometimes he was
thus compelled to do things which seemed strange even to him.
When, in 1865, little Katie Mills died, he had to act the part of
undertaker. He said:

It fell to me to make the coffin, which I did as well as I could

from memory. I could not tell the carpenter, and I had to do
the work myself. He did the rough work, and I did the cutting
and fitting. I had to go entirely by my eye, and I found it no
easy matter to get it in every respect in proportion. We
covered it with black velvet outside, and inside with white
 linen. It looked very well when finished, and pleased Mr. and
Mrs. Mills very much. It is a work I never thought of doing.

At one point on the way through Siberia when homeward bound

on his last furlough the train was halted by some defect in the
working of the mechanism of the locomotive. Dr. Mateer, on account
of the delay, got out of his compartment and went to see what was
the matter. He saw that the locomotive was a huge Baldwin, with
whose construction he had familiarized himself when in the United
States on a previous furlough, and he quickly discovered the cause
of the trouble. He could speak no Russian, and the men in charge of
the engine could speak no English, but he managed to show them
the cause of the defective working of the mechanism, and how to
remedy it; and soon the train was again speeding on its way.
The time never came during his long residence in China when a
necessity did not occasionally force itself on him to utilize his
mechanical gifts, and not infrequently on the common utensils of life.
In Wei Hsien he often spent hours directing in such repairs as were
needed for furnaces and the like.
Few of his later and larger achievements in this field could be fairly
regarded as works of necessity, strictly speaking; they rather were
meant to be aids in the great enterprise of evangelizing the Chinese
Empire. He was thoroughly convinced that one of the most powerful
agencies that could be employed for this purpose was the school
and the college. He was equally sure that of all the studies that could
be introduced into the curricula of these institutions, none could be
so effective in opening the way for the gospel as that of the natural
sciences, and especially physics, inclusive of modern mechanical
appliances of its principles. He believed that if bright young men
were educated in that kind of knowledge, and sent out under
Christian influences among their own people, if they were also
converted to Christianity, the outcome must be the dissipation of the
existing blind adherence to the superstitions and ideas of centuries
long remote in the past; and that with this must come the opening
wide of the door for the entrance of Christianity. That was his
forecast; and the present situation in China goes far toward
vindicating the wisdom of it. But to teach effectively the natural
sciences he must have apparatus. The only way he could secure this
was by buying what he could, and by utilizing his own ability to set
this up, and to add as much as possible for the outfit yet needed.
Such was the prime object not only of what in a more limited sense
constituted the apparatus of the school and college, but also of such
larger appliances as the plant for heating and lighting the premises.
These were far more than conveniences that helped to better work;
they were themselves constant exhibitions to the students and to the
people at large of the principles of natural science, and of their value
in the affairs of actual life.
Dr. Mateer utilized his outfit of apparatus and machinery as a
means of reaching others besides the students in his own institution,
with the influence of modern science, thus opening a way into their
minds for the gospel. As to one of his methods of accomplishing this
object Ada gives a graphic account:

At the time when the official examinations were held in

Tengchow, a large number of scholars came to town, hoping
to secure a degree, which should be the first step toward
official preferment. So many of these, having heard the fame
of the foreign machine, came to see and to hear, that Dr.
Mateer used to give up his time to them during the days they
were at leisure. Finally the opportunity to do good in this way
proved so great that a place was provided for the purpose,
which was also much used at the Chinese New Year, when all
the town and countryside give themselves up to recreation.
After the “Mandarin Lessons” began to bring in money, he
devoted the profits to the building of a large museum, with an
entrance on the street. One half was a big audience room, so
arranged that it could be darkened down for stereopticon or
cinematograph exhibitions. But it usually served as an
audience room, where the crowds could sit and listen to
preaching, while the detachment that preceded them was
shown through the inner room by expert assistants. What a
chamber of wonders that inner room proved to them! Here
 was a man, using a single hand to turn a small crank, grinding
corn as fast as a woman or a donkey could do it on the
millstones with much more labor. Here in cases were birds
stuffed, and on the walls pictures of strange animals. Here
was a man turning a large crank that in some mysterious way
made a little iron car overhead first send out sparks, and then
run all around the room on a circular railroad. They wondered
if it would not have been easier for the man to drag the car
around on the ground! There was an oil engine at the end of
the room, that was a wonder, no mistake; and a “shocking”
machine that shocked them indeed; and untold other
wonders. When the tour of the room was finished, the crowd
was let out by another door, their almond eyes quite round,
while a signal given by a steam siren showed it was time for
the next group to go in, and “open-open-eyes,” as they call
sight-seeing.

Occasionally a mandarin of high order came to witness the

marvels. The report of the Shantung Mission for 1909 says that
through the agency of the chapel and museum twelve thousand
people were brought into touch with the gospel during the year; so
the work still continues.
Another good account to which Dr. Mateer turned this peculiar gift
was that of starting industries for native Christians and promoting
self-help among the needy. Now it was a loom for weaving coarse
Chinese linsey or bagging, or a spinning or a knitting machine, that
he ordered; again, he inquired for a roller press to be used for drying
and pressing cotton cloth after dyeing; and more than once he sent
for a lathe for a Chinese blacksmith. In 1896 he interested himself in
procuring an outfit for a flouring mill. He said: “The enterprise of
starting the mill was conceived by Chinese Christians, and they are
going to form a company to raise the money. I do not think that there
is a roller mill in China,—certainly not in north China.... We
personally will not make a cent out of it; but we are interested to get
the Chinese Christians started in an enterprise by which they can
make a living, and introduce improvements into their country.”
 His apprentices went out in many instances master blacksmiths,
machinists, and electricians, and had no difficulty in finding places. A
Chinese general temporarily at Tengchow employed one of these
men as a blacksmith, and his order was so evidently filled according
to western methods that he paid a visit to the wonderful shop of this
wonderful master. The very last man for whom he obtained a place
was his most skilled electrician and his latest foreman. This man
started a shop up at the capital of the province, and for its outfit Dr.
Mateer carried on an extensive correspondence and procured large
invoices of goods. Because of the provincial university established
there under the new educational régime there was imperative need
of such an establishment, and the outlook for success was excellent.
Unfortunately for the proprietor, however, the Chinese officials were
equally alive to the opportunity and were jealous of a rival. So they
managed to compel him to sell out, though they broke the fall a little
for him by retaining him as foreman. It is said that the thought of this
workman’s troubles lay heavy on the heart of Dr. Mateer in his last
illness. It was usually for the poor that he interested himself after this
practical fashion; yet he did not refuse to lend aid to others in
promoting enterprises that would be of general advantage. For a
wealthy Chinaman who owned a coal mine that had been flooded
with water he went to a great deal of trouble in order to put him in the
way of securing a suitable pump. But whether it was for rich or poor
that the opportunity came to render such services, he put aside all
thought of his own ease or name or profit, and did the best in his
power.
He had special satisfaction in the manufacture of electrical
machines, though it was no easy matter to cut and bore the large
glass wheels without breaking them, and to adjust all parts so that
the greatest efficiency was attained. Ada says:

When a machine was perfected, giving an unusually long

spark, he always liked to take me over at night to the shop to
see it perform. I well remember the last time,—at Wei Hsien.
At one end of the shop was the windmill. Here he stopped to
show me a way of equalizing the stroke of the windmill pump
 piston, by hanging on an old kettle of scrap iron. Then he took
me into an inner room, where on one end of a long table
stood the newly finished machine,—a beauty, no mistake.
Having forgotten some necessary key, he took the lantern and
went to get it, leaving me in the dark. I noticed sounds, the
dripping of water in the well; but what was the ticking I heard?
On the return of the lantern I saw the cause,—a number of
clock dials all hung on the wall, and all to be run by one clock
by means of electricity. These were for the college recitation
rooms when they should be finished. Then Calvin made the
new machine do its work. Adjusting carefully the mechanism,
and then measuring the spark, he exclaimed with boyish glee,
“There, isn’t that a beauty!”

Dr. W. A. P. Martin, of Peking, related in the “Chinese Recorder” of

December, 1908, this incident as to Dr. Mateer: “It was once my
privilege to spend part of a vacation in his hospitable home at
Tengchow. I found him at work constructing scientific apparatus with
his own hands and wrestling with a mathematical problem which he
had met in an American magazine. When I solved the problem, he
evinced a lively satisfaction, as if it were the one thing required to
cement our friendship.” The problem was to find the diameter of an
auger, which, passing through the center of a sphere, will bore away
just one half of its bulk. It is easy to see that to a man of that sort his
work and the scientific and practical problems constantly arising in
connection with the making of apparatus and the adjustment of
machinery must have been in themselves a rich source of pleasure,
though he never allowed himself to be so fascinated by his shop as
to break in on what he conceived to be his higher work. Speaking of
his last years, Ada says: “He would go out wearied with the baffling
search for a way of expressing clearly in Chinese a thought none too
clear in the original Greek, his forehead grooved with the harrows of
thought. He would come back from the shop an hour later, with well-
begrimed hands, a new spot on his long Chinese gown, a fresher
pink in his cheeks, a brighter sparkle in his eyes, and his lips parted
with a smile. Then, having washed, he would immediately set himself
again to the work of revision.”
 He loved also to share this joy, so far as it could be done, with
others. At the Synod of China with his apparatus he gave several
exhibitions that were greatly appreciated. At Wei Hsien he rendered
similar services in the high schools, and at Chefoo in the school for
the children of missionaries. The Centennial Fourth of July, being
quite an exceptional occasion, he celebrated not with ordinary
gunpowder, but by setting off a considerable quantity of detonating
chemicals. In the early days at Tengchow a home-made electric fly
whisk whirled above the dining table, and a little pneumatic fountain
playing in a bell glass rendered the room and the meals additionally
pleasant to the family and to the guests. Ada writes:

But the thing that most of us will remember longest is an

illustrated lecture on electricity delivered to the college in Wei
Hsien, and afterward to the foreigners there. As we sat in a
darkened room in the college watching the long sparks of fire,
the twisting circles of many-colored light, half illuminating a
tall, white-bearded figure in a long black gown, he seemed to
us like some old magician, learned in the black arts, now
become bright arts, invoking to his aid his attendant spirits.
Nor was the enchantment diminished when afterward, more
wonderful than a palmister, he showed us by the x-rays the
bones of our hands. A few weeks later one of the ladies of the
compound gave an evening entertainment in which each one
in the station was hit off in some bright way, and we were to
guess the name. One number of the programme was this: A
black-robed figure with cotton beard appeared, leading a
youth whom he seated in a chair. Then the venerable
personage proceeds to examine the head of the stripling with
a stereoscope covered with black cloth, supposed to be a
fluoroscope, while an alarm clock in a tin pail near by supplies
the crackling of electricity. He gives a careful examination,
shakes his head, and pronounces the verdict in one word,
“Empty.” In explanation of the tableau it only needs to be said
that between the exhibition and the entertainment Dr. Mateer
had given the young men of the station their examination in
the language.
 XIII
THE MANDARIN VERSION

“I am mortgaged to the Bible revision work.... It cost me a great effort to

engage in it, but it will probably be the most important work of my
life.”—letter to secretary brown, June 13, 1896.

To tell this part of the story of Dr. Mateer’s life satisfactorily, I must
begin with the first general missionary conference, held at Shanghai
in May, 1877. For two years previous he had served on a committee
to prepare the way for the meeting, and in this capacity he had
rendered much valuable assistance. At that conference he read a
paper in which he elaborately discussed the subject of “The Relation
of Protestant Missions to Education.” The meeting was regarded as
successful, and a second was called, to assemble at Shanghai, in
May, 1890. It was at this conference that the movement for a revision
of the Bible in Chinese took actual measures toward realization.
For the sake of any readers not well informed as to the Chinese
language, a few preliminary statements concerning it may be
desirable here. In a very broad and general sense it may be said that
as to elements, one tongue prevails throughout China proper; but
that there is also much important variation in this general tongue.
First of all, it needs lo be noted that the language takes on two
principal forms,—the classic, or Wen-li, and the spoken, or
Mandarin. The classic has come down through the centuries from
the times of Confucius and Mencius, and remains comparatively the
same as it is found in the writings of those sages. This is accepted
as the model for all writing; and for that reason Chinese students
have been required to spend the greater part of their time in
memorizing those ancient books, so that they might not only absorb
their teaching, but also especially that they might be able to
reproduce their style. The classic Chinese is stilted and so
condensed that in comparison with it a telegram would seem diffuse;
and though many of the characters are the same as those used in
writing the spoken language, yet the meaning and often the sound of
characters is so different that an illiterate person would not
understand it on hearing it read. The spoken language, on the other
hand, may be compared with English as to its use. Good English is
very much the same throughout the countries where it is the
vernacular, and though it takes on local dialects, it remains
everywhere intelligible. So, broadly speaking, is it also as to the
spoken Chinese in a large part of the empire. From the Yangtse up
into Manchuria, though the pronunciations differ very much, the
colloquial if put into writing is understood. In other words, with
differences of dialect and pronunciation it is the speech of perhaps
three hundred millions of people. The regions excepted lie along the
coast from Shanghai down, and inland south of the Yangtse, where
the distinct tongues are numerous and are largely unintelligible
except in their own localities.
It has been the rule in China that a mandarin must not be a native
of the province where he holds office; and, of course, it is essential
that he should be acquainted with the speech which constitutes the
lingua franca. Perhaps for this reason it is called Mandarin. But down
to the time when missionary publications rendered it common in
print, it was not employed in that mode. All books, business or
government documents, the one newspaper of the country, which
was the court gazette, and all letters were in the higher or, as it is
called, the Wen-li form, the only exception being some novels, and
even these were streaked with Wen-li. This, however, ran through
gradations,—from the highest, which is so condensed and so bristles
with erudite allusions that only a trained scholar can understand it,
down to a modification which is so easy that with a slight alteration of
particles it is almost the same as the Mandarin.
During the long period of the nineteenth century preceding the
meeting of the second general missionary conference, a number of
translations of the Scriptures, some of them of the whole, and some
of parts, had been made, and had come more or less into use. The
men who did this pioneer work deserve to be held in perpetual
esteem, especially in view of the difficulties under which they
labored. Among the missionaries who sat in that conference there
was no disposition to withhold this honor, or to disparage the value of
these early translations; but there was so widely prevalent among
them and their associates at that time on the field a conviction that
no existing version was satisfactory, that they recognized it as a duty
to take up the subject, and to initiate steps looking to the production
of a better. An informal consultation as to this was held by a few
men, a couple of days before the conference assembled; but
inasmuch as Dr. Mateer had not been invited, he did not attend.
Another consultation was held the following day, and because of his
great interest in the subject of a Bible revision, he attended without
an invitation. The views expressed clearly indicated that there was a
general agreement that a revision was desirable, but it also was
made very plain that beyond this there was a wide divergence of
opinion. We will allow one of his letters to a representative of the
American Bible Society, under date of May 26, 1890, to tell the next
step in this great undertaking:

As I walked home from the meeting, and revolved in my

mind the difficulty of the situation, the idea of an executive
committee, to whom the whole work should be intrusted,
came across my mind. When I reached my room I sat down,
and in a few minutes and without consultation with anyone,
wrote out the plan, which without essential modification was
subsequently adopted. It seemed to strike all parties very
favorably. On the second day of the conference two large,
representative committees were appointed by the conference,
one on Mandarin and one on Wen-li. I was a member of both
these committees. Each committee had a number of
meetings, in which the subject was freely and fully discussed
in all its bearings. It was evident that there was a general
desire for a version in simple Wen-li, and, the difficulties being
less in regard to the work already done, a conclusion was first
reached in regard to this version. In Mandarin the difficulties
were greater.
 An agreement, however, was reached. The version in the higher
classic style then gave the most trouble, but a satisfactory basis for
this also was agreed upon; and the reports as to all three versions
were adopted unanimously by the conference. In the same letter he
says: “I worked hard for these results, and felt no small satisfaction
in seeing such perfect unanimity in the adoption of the plan
proposed. I have never done anything in which I felt more the
guiding hand of God than in drawing up and carrying through this
plan.”
The selection of translators for each of the projected new versions
was handed over respectively to executive committees; and Dr.
Mateer was appointed on that having charge of the Mandarin, and
made chairman of it. He heard that he was talked of as one of the
revisers for that version, but as yet he had not decided what was his
duty, if chosen. It will again be best here to take up from one of his
letters the thread of the narrative. Under date of December 13, 1890,
he writes to Dr. Nevius:

I can truly say that before I went to the conference I never

even dreamed of what has come to pass. It never occurred to
me, before the conference, that I should take any prominent
part in the matter of Bible translation. I felt that education was
the only field in which I should come to the front. I was never
in my life so providentially led as I was in this matter. I was
selected chairman of the Mandarin Executive Committee and
have been pushing the getting of translators. The first few
months were spent in corresponding and comparing notes as
to men. We took a ballot recently, which resulted in the
election of five, ... I being the only one who received a
unanimous vote. We are now voting for the others, to make
up the seven.... My book of “Mandarin Lessons” has no doubt
brought me forward, and its preparation has in a measure
fitted me for the work. My personal preferences are against
the work of translation, and I would fain decline it, but I don’t
see how I can in view of the circumstances. I feel my
incompetency, especially in Greek and Hebrew, and you may
 be sure I am very loath to give up the educational and literary
work on my hands. Much of it is half finished. But if the
Mandarin Bible is to be made, some one must do it;
moreover, the men who do it must have the confidence of the
missionary body; otherwise it will be a failure. As it is,
circumstances have led me to the position, and the strong
opinion of the men on the committee, and of others, leads me
to feel that I cannot lightly refuse.

In November, 1891, the revisers met at Shanghai. Dr. Mateer, in a

letter written in the following January, said:

The scheme for the revision of the Chinese Bible set on

foot by the conference is now fairly organized, and approved
by the three great Bible societies. The work of pushing the
organization has fallen largely on me, and I feel no small
sense of relief now that it is successfully accomplished.
Contrary to my own desire, I am compelled to lake a share as
one of the revisers in Mandarin; not that I do not relish the
work, but because it will of necessity interfere with many of
my cherished plans. We had a meeting of all the revisers of
the three versions, and it was a fairly harmonious and an
altogether successful meeting. A great work is before us
which I trust we may, in the good providence of God, be
enabled to accomplish.

The interval of about a year and a half between the general

conference and the organization just mentioned was required
because of the difficulty of selecting and securing the translators.
These for the Mandarin version, as that body was originally
constituted, consisted of Henry Blodgett, George Owen, Chauncey
Goodrich, J. R. Hykes, Thomas Bramfitt, J. L. Nevius and C. W.
Mateer. During the years in which this work was continued there
were in the membership so many changes caused by death, removal
and other causes, that Dr. Goodrich and Dr. Mateer alone continued
from the beginning until the translation of the New Testament, the
part of the Bible first revised, was tentatively completed. Mr. Baller of
the China Inland Mission stands next in length of service, having
joined the committee in 1900. Dr. Mateer in the work of revision had
the assistance of two Chinese Christians whose services were so
large and valuable that they deserve more than a passing mention
here. In a recent letter Dr. Goodrich pays them the following just
tribute:

Dr. Mateer, in the work of rendering the Scriptures into a

universal Mandarin colloquial, had two exceptionally fine
teachers. The first was Mr. Tsou Li Wen, an ordained pastor,
who left his parish to engage in this work. Mr. Tsou was
trained by Dr. Mateer in his college, receiving his theological
training under Drs. Nevius, Mateer and others. He was a man
of beautiful spirit, discriminating mind, and a fine sense of
language. He was also a man of indomitable perseverance.
After a strenuous day’s work of eight hours or more, he would
often toil by himself far into the night, seeking for some
phrase or phrases which expressed more exactly or more
beautifully the meaning of the original. And before the final
review, both he and my own lamented teacher (Chang Hsi
Hsin) would bestow the greatest pains, in the hours when
they should have been sleeping, in a careful inspection of the
work. Thus did Mr. Tsou toil, while separated from his family
for long periods of time; his work on Bible revision being as
truly a labor of love as that of any member of the committee.
But alas! Mr. Tsou’s life burned out all too soon in his
exhausting labors. But how I should like to see his crown, and
his shining face!
Happily for the work, Dr. Mateer had another scholar,
trained also in his school, Mr. Wang Yuan Teh, a young man
of keen, incisive, logical mind, who had read all the best
books in the Mandarin colloquial. Mr. Wang was quick to see
any fault in the structure of a sentence, and insistent on its
being put right. He also worked most faithfully in this
translation, refusing offers which came to him of a salary
 several times the amount he received. I think he was held,
partly by Dr. Mateer’s personality, which drew him strongly,
and partly by his own love for the work itself. When the chariot
of fire came for Dr. Mateer, he left us, much to our regret and
loss.
The work of these two men has entered largely into the
present translation of the New Testament, and the influence of
their work, as of Dr. Mateer’s, abides, and will continue to be
felt, till the great work of rendering the Bible into a universal
Mandarin is finished.

Dr. Mateer himself, in the preface to his “Mandarin Lessons,”

makes acknowledgment of the valuable services rendered in the
preparation of that work by Tsou Li Wen, and also by his own wife.
The Mandarin Committee, at the meeting in 1891, after
organization, proceeded to divide up the books of the New
Testament among themselves for work, and adopted a plan of
procedure. Each man was first carefully to revise or translate his own
portion; and then to send it around to the others, who were to go
over it, and write their suggestions of emendations, each in a column
parallel to the proposed text. Next, the original translator was to take
these emendations, and with their help was to prepare a text in
Mandarin for submission to the entire committee. Broadly speaking,
this was the method pursued to the end, though with some
modifications compelled or suggested by experience. It was hoped
that comparatively rapid progress would be made; but in reality the
committee did not come together again until September, 1898; and
even then, only the Acts of the Apostles was ready for general
revision. For this delay there were various causes, such as the death
of Dr. Nevius and the resignation of others, and the absence of Dr.
Mateer on furlough home; but the chief cause was that every
member was burdened with so much other work that only a fraction
of his time could be given to this duty. Dr. Mateer, for example, found
himself loaded down with other literary and missionary labors. At the
meeting held at Tengchow, in 1898, he was elected chairman of the
committee. This was an honor, but it also carried with it peculiar
duties which materially added to his burden. The committee could
muster only five members for that sitting, but they proceeded with
their work, and at the end of two months and a half they finished the
book of Acts; and then they separated.

MANDARIN REVISION COMMITTEE AT WORK

DR. S. LEWIS DR. DR. MATEER M. BALLER

(American Methodist GOODRICH (Presbyterian) (China Inland
Episcopal) (Congregational) Mission)

That meeting by actual experience brought out distinctly not only

the difficulties of necessity arising from the translation of particular
books of the Bible, and indeed of every verse; but also others of a
more general character, some of which had previously been more or
less clearly seen. Should the new version take as its basis one or
more of the translations already in existence; or should it go back
straight to the original Greek, and use the existing translations
merely as helps? In any case, constant reference to the original was
a necessity. For this, which of the published texts should be
accepted as the standard? The meeting also disclosed a wide
divergence of opinion as to the style of Mandarin that ought to be
employed. On that subject in 1900, Dr. Mateer expressed himself
fully and strongly, in an article published in the “Chinese Recorder.”
He said:

The Mandarin Bible, in order to fulfill its purpose, should be

such as can be readily understood by all when heard as read
aloud by another. The fundamental distinction between Wen-li
and Mandarin is that the former is addressed to the eye, the
latter to the ear. In all Protestant churches the reading of the
Scriptures has, from the first, constituted an important part of
public worship. In order that this reading may serve the
purpose intended, the Scripture must be so translated as to
be intelligible to the common people. Only thus will they hear
it, as they did its Author, “gladly.” It is not enough that those
who know “characters” should be able to read it intelligently,
but rather that those who do not know “characters,” and who
in fact constitute by far the greater part of the Chinese people,
should be able to understand it when it is read to them. Here
then is the standard to be aimed at,—a version that
represents the Chinese language as it is spoken, and
addresses itself to the ear rather than to the eye.

He summarized the chief characteristics of the proper style thus:

that words should be employed which the people who commonly use
Mandarin can understand; that sentences should conform to the
model of the spoken language; and, concerning both of these
requisites, that such care should be taken as to brevity, the order of
words and clauses, the connective particles, and the evident
movement of thought as expressed, that the Chinese would
recognize in it a people’s book; and yet one that is free from
undignified colloquialisms and localisms. All this he held up as an
ideal, not likely to be fully realized by any set of translators, but if
distinctly aimed at, more sure to be nearly approached. Toward the
close of the work on the Mandarin version still another question of a
general nature arose. Throughout most of their labors the committee
had before them the revised easy Wen-li translation, and for a part of
the time they also had the revised classic Wen-li Bible. Ought the
three revised Chinese versions to be harmonized, so as to eliminate
all variations? That, of course, would be ideal. On this question the
report of the Mandarin Committee, which was as to substance
prepared by the chairman, took the negative. It said:

The differences are not great, and where they exist, the
versions will serve Chinese students as a sort of commentary.
There are a multitude of questions in Biblical interpretation
which no translation can settle once for all. Moreover, ninety-
nine out of a hundred of those who use the Mandarin will
never look at any other translation. Two versions in perfect
accord seem like a fine product, but it is difficult of realization.
An attempt at reconciling the present versions would develop
many difficulties. A Mandarin sentence especially is not easy
to tamper with. The change of a single word would often
dislocate a long sentence, and necessitate retranslation and
adjustment to the context.

The Mandarin committee of translators continued their tentative

revision of the New Testament until late in 1906, a period of fifteen
years, counting from the date of their first meeting for organization
and assignment of specific duties. They held eight different sessions,
being almost one each year after they were ready with actual work;
and none of the sessions were shorter than two and a half months,
and one of them stretched out to six months. They assembled at
Tengchow, near Peking, at Shanghai, and most frequently at Chefoo.
In the final report is the record: “The chairman can say for himself
that he has given the equivalent of about seven years all-day labor to
this work. He was present at every meeting, and first and last missed
but one day’s session.” Each of the meetings took on distinctive
incidental associations. The third was held at Shanghai, and from
December, 1900, ran over some months into 1901. At that time, on
account of the Boxer uprising, missionaries were temporarily there
as refugees from all the provinces directly concerned in the version.
The sittings were in a small upper room in the Union Church, which
came to be called “the Jerusalem Chamber,” and visitors were many.
They saw two rows of men, one on each side of a long table, yellow
faces being sandwiched alternately with white, as each translator
had, as usual in this work, his Chinese assistant at his side. Often
the discussions were carried on in Mandarin, so that these assistants
might be able to understand and pass their opinion. Incidentally it
may be noted that besides his work on the revision, Dr. Mateer often
met with the refugee missionaries during this period and greatly
gratified them by participating in the discussion of practical problems.
After the Mateers returned from their furlough, the sessions were
all held at Chefoo, first in one of the rooms of the China Inland
Mission Sanitarium, and later in a large upper room in the Missionary
Home, overlooking the bay. Usually at the commencement of their
meetings they sat together for three hours in the morning, and
reserved the rest of the day for such private study as they wished to
make; but as the time wore on they would increase the sittings to as
many hours also in the afternoon, and crowd the private review into
such odd moments as were left. To anyone, these protracted labors
on such a work must have become exceedingly tedious and almost
irksome; but to no one was it more so than to Dr. Mateer. He knew
Mandarin almost as if it had been his native tongue; but the
Mandarin which he knew had often to be modified and expressions
adjusted, so that a Scripture written in it would suit other regions of
China as well as those with which he was familiar. In writing his
“Mandarin Lessons” and in preparing his educational books he had
only to ascertain to the best of his ability how to express his ideas in
Chinese, and that was the end of the search; but here he had to do
his best, and then submit his product to the opinion of others, and
often with the result of changes which did not commend themselves
to his preference. Yet, on his return home from the sittings he would
say: “I ought not to complain. I get my way oftener than any other
man does. Only I cannot help thinking of the work I have laid aside
unfinished in order to do this.” After each meeting the year’s work
was printed, marked “Tentative Edition,” and with a slip inviting
criticism was sent to the missionaries in north China and Manchuria.
These criticisms were all to be canvassed before the edition could be
printed that was to be presented to the Centenary Conference, to
which they were to report.
The final meeting for the tentative revision of the New Testament
lasted for more than five months, and the work was pushed with
even more than the usual vigor. The Centenary Missionary
Conference for China was only a year ahead when they began. After
the conference the revision was to run the gauntlet of criticisms, and
these were to be canvassed; and thus at last the revision was to take
its permanent form. Mrs. Mateer gives the following graphic account
of one of the closing incidents of that session.

Passage had already been engaged for the Goodrich family

on a steamer sailing north. The baggage was all carried
down, the family all waited on the upper veranda, with hats
on, and the Doctor’s hat was ready for him to seize as soon
as he should get out of the meeting. The “rickshaw” men were
waiting, ready to run with their loads. But still no sound of
approaching feet! Finally, as it got dangerously near the hour
of sailing, Mrs. Goodrich said, “I must go and hurry them up.”
So she marched boldly down the hall, listened a minute at the
door, and came back with her fingers on her lips. “Those dear
men are praying,” she whispered; and tears filled our eyes as
our hearts silently joined in the prayer. Of course, every
morning session was opened with prayer; but this was the
consummation of all these years of toil, the offering of the
finished work at the altar.

Although the committee completed their revision at that session,

so far as this was possible until the conference should meet and
approve or disapprove it, there was very considerable work of a
tedious nature left to Dr. Mateer to perform. The finishing touches yet
to be put upon portions of the version were not a few; but the thing
that required of him the most protracted and delicate attention was
the punctuation. For this he introduced a new system which seemed
to him to be best for the Chinese language, and which can be
estimated fairly only by a scholar in that tongue. To him also as