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Methods in
Molecular Biology 2630
Tamas Dalmay Editor
MicroRNA Detection
and Target
Identification
Methods and Protocols
Second Edition
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MicroRNA Detection and Target
Identification
Methods and Protocols
Second Edition
Edited by
Tamas Dalmay
RNA Biology, University of East Anglia, Norwich, UK
Editor
Tamas Dalmay
RNA Biology
University of East Anglia
Norwich, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
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Preface
This book is a second edition of a previous book in this series; therefore, it is unnecessary to
introduce microRNAs (miRNAs) to any reader who is reading this preface. The previous
book (published in 2017) described protocols to detect, profile, and manipulate miRNAs in
various organisms, as well as how to predict and validate targets of miRNAs in plants and
animals. However, a lot of new techniques have been developed in the last 5–6 years and
existing techniques evolved, which warranted a second edition.
The first chapter gives an overview of the detection methods, and the second chapter
describes how to identify and validate microRNAs. Chapters 3, 4, 5, 6, and 7 cover different
protocols for microRNA detection, some of them are revised versions from the first edition
and some are new. Chapters 8, 9, and 10 provide protocols for different approaches to
profile the expression level of microRNAs. A new chapter in the book is a protocol for spatial
expression analysis (Chap. 11). Chapters 12, 13, and 14 describe in silico analysis of
microRNAs and their targets. Finally, the last two chapters give protocols for functional
analysis of microRNAs (Chap. 15) and their targets (Chap. 16) by CRISPR/Cas.
Norwich, UK Tamas Dalmay
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Detection of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Afrah Bawazeer and David C. Prince
2 MicroRNA Identification, Target Prediction, and Validation
for Crop Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Vrantika Chaudhary, Sumit Jangra, Apurva Mishra, and Neelam R. Yadav
3 MicroRNA Detection with CRISPR/Cas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Xinyuan Qiu, Chuanyang Liu, Chushu Zhu, and Lingyun Zhu
4 Detection of MicroRNAs by Northern Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Claudia Martinho and Sara Lopez-Gomollon
5 MicroRNA Detection at Femtomolar Concentrations with Isothermal
Amplification and a Biological Nanopore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Sotaro Takiguchi and Ryuji Kawano
6 Detection of MicroRNA Expression Dynamics Using
LNA/DNA Nanobiosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Yuwen Zhao and Shue Wang
7 Programmable Ultrasensitive Molecular Amplifier for Digital
and Multiplex MicroRNA Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Yannick Rondelez and Guillaume Gines
8 Small RNA Profiling by Next-Generation Sequencing Using
High-Definition Adapters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Rocky Payet and Martina Billmeier
9 Quantification of MicroRNAs or Viral RNAs with Microelectrode
Sensors Enabled by Electrochemical Signal Amplification . . . . . . . . . . . . . . . . . . . . 117
Sarah Ake, Swagatika Kamila, and Gangli Wang
10 Discovery and Evaluation of Extracellular MicroRNA Biomarkers
in Plasma, Ascites, and Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Luděk Záveský and Ondřej Slanař
11 Determining miRNA Expression Patterns in Xenopus . . . . . . . . . . . . . . . . . . . . . . . 145
Marco Antonaci, Alice M. Godden, and Grant N. Wheeler
12 Overview of Computational and Experimental Methods to Identify
Tissue-Specific MicroRNA Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Ulf Schmitz
13 sRNAtoolbox: Dockerized Analysis of Small RNA Sequencing
Data in Model and Non-model Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Cristina Gomez-Martı́n, Ernesto Aparicio-Puerta, and Michael Hackenberg
vii
viii Contents
14 MicroRNA–Target Identification: A Combinatorial

In Silico Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
K. M. Taufiqul Arif, Rachel K. Okolicsanyi, Larisa M. Haupt,
and Lyn R. Griffiths
15 An Efficient CRISPR-Cas9 Method to Knock Out MiRNA
Expression in Xenopus Tropicalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Alice M. Godden, Marco Antonaci, and Grant N. Wheeler
16 Interrogation of Functional miRNA-Target Interactions
by CRISPR/Cas9 Genome Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Qianxin Wu, Yale S. Michaels, and Tudor A. Fulga
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Contributors
SARAH AKE • Department of Chemistry, Georgia State University, Atlanta, GA, USA
MARCO ANTONACI • School of Biological Sciences, University of East Anglia, Norwich, UK
ERNESTO APARICIO-PUERTA • Chair for Clinical Bioinformatics, Saarland University,
Saarbrücken, Germany
TAUFIQUL K. M. ARIF • Centre for Genomics and Personalised Health, Genomics Research
Centre, School of Biomedical Sciences, Queensland University of Technology (QUT), Kelvin
Grove, QLD, Australia
AFRAH BAWAZEER • School of Biological Sciences, University of East Anglia, Norwich, UK
MARTINA BILLMEIER • Institute of Medical Microbiology and Hygiene, University of
Regensburg, Regensburg, Germany
VRANTIKA CHAUDHARY • Department of Molecular Biology, Biotechnology, and
Bioinformatics, CCS Haryana Agricultural University, Hisar, India
TUDOR A. FULGA • Vertex Pharmaceuticals, Boston, MA, USA
GUILLAUME GINES • Gulliver Laboratory, ESPCI Paris – Université PSL, Paris, France
ALICE M. GODDEN • School of Biological Sciences, University of East Anglia, Norwich, UK
CRISTINA GÓMEZ-MARTÍN • Department of Pathology, Cancer Center Amsterdam,
Amsterdam UMC, VU University, Amsterdam, The Netherlands
LYN R. GRIFFITHS • Centre for Genomics and Personalised Health, Genomics Research
MICHAEL HACKENBERG • Faculty of Science, and Bioinformatics Laboratory, Genetics
Department, Biomedical Research Centre (CIBM), Universidad de Granada, Granada,
Spain; Excellence Research Unit “Modeling Nature” (MNat), Instituto de Investigacion
Biosanitaria ibs. GRANADA, University Hospitals of Granada-University of Granada,
Granada, Spain
LARISA M. HAUPT • Centre for Genomics and Personalised Health, Genomics Research
SUMIT JANGRA • Department of Molecular Biology, Biotechnology, and Bioinformatics, CCS
Haryana Agricultural University, Hisar, India; Advanced Centre for Plant Virology,
Division of Plant Pathology, ICAR- Indian Agricultural Research Institute, New Delhi,
India
SWAGATIKA KAMILA • Department of Chemistry, Georgia State University, Atlanta, GA, USA
RYUJI KAWANO • Department of Biotechnology and Life Science, Tokyo University of
Agriculture and Technology, Tokyo, Japan
CHUANYANG LIU • Department of Biology and Chemistry, College of Liberal Arts and
Sciences, National University of Defense Technology, Changsha, Hunan, China
SARA LOPEZ-GOMOLLON • Department of Plant Sciences, University of Cambridge,
Cambridge, UK
CLAUDIA MARTINHO • Department of Plant Sciences, University of Cambridge, Cambridge,
UK; Department of Algal Development and Evolution, Max Planck Institute for Biology
Tübingen, Tübingen, Germany
ix
x Contributors
YALE S. MICHAELS • Stem Cell Bioengineering Lab, School of Biomedical Engineering, The
University of British Columbia, Vancouver, BC, Canada
APURVA MISHRA • Department of Molecular Biology and Genetics, Arsuaga-Vazquez Lab,
University of California, Davis, CA, USA
RACHEL K. OKOLICSANYI • Centre for Genomics and Personalised Health, Genomics Research
ROCKY PAYET • School of Biological Sciences, University of East Anglia, Norwich, UK
DAVID C. PRINCE • School of Biological Sciences, University of East Anglia, Norwich, UK
XINYUAN QIU • Department of Biology and Chemistry, College of Liberal Arts and Sciences,
National University of Defense Technology, Changsha, Hunan, China
YANNICK RONDELEZ • Gulliver Laboratory, ESPCI Paris – Université PSL, Paris, France
ULF SCHMITZ • Department of Molecular & Cell Biology, College of Public Health, Medical
& Vet Sciences, James Cook University, Douglas, QLD, Australia; Centre for Tropical
Bioinformatics and Molecular Biology, Australian Institute of Tropical Health and
Medicine, James Cook University, Cairns, QLD, Australia
ONDŘEJ SLANAŘ • Institute of Pharmacology, First Faculty of Medicine, Charles University,
Prague, Czech Republic; General University Hospital in Prague, Prague, Czech Republic
SOTARO TAKIGUCHI • Department of Biotechnology and Life Science, Tokyo University of
Agriculture and Technology, Tokyo, Japan
GANGLI WANG • Department of Chemistry, Georgia State University, Atlanta, GA, USA
SHUE WANG • Department of Chemistry, Chemical and Biomedical Engineering, Tagliatela
College of Engineering, University of New Haven, West Haven, CT, USA
GRANT N. WHEELER • School of Biological Sciences, University of East Anglia, Norwich, UK
QIANXIN WU • Sanger Centre, Hinxton, Saffron Walden, UK
NEELAM R. YADAV • Department of Molecular Biology, Biotechnology, and Bioinformatics,
CCS Haryana Agricultural University, Hisar, India
LUDĚK ZÁVESKÝ • Institute of Biology and Medical Genetics and Institute of Pharmacology,
First Faculty of Medicine, Charles University, Prague, Czech Republic; General University
Hospital in Prague, Prague, Czech Republic
YUWEN ZHAO • Department of Chemistry, Chemical and Biomedical Engineering,
Tagliatela College of Engineering, University of New Haven, West Haven, CT, USA;
Department of Bioengineering, Lehigh University, Bethlehem, PA, USA
CHUSHU ZHU • Graduate School, National University of Defense Technology, Changsha,
Hunan, China
LINGYUN ZHU • Department of Biology and Chemistry, College of Liberal Arts and Sciences,
National University of Defense Technology, Changsha, Hunan, China
Chapter 1
Detection of miRNAs
Afrah Bawazeer and David C. Prince
Abstract
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression. They play an important
role in many biological processes including human diseases. However, miRNAs are challenging to detect
due to their short sequence length and low copy number. A number of conventional (e.g., Northern blot,
microarray, and RT-qPCR) and emerging (e.g., nanostructured materials and electrochemical methods)
techniques have been developed to detect miRNA, each with their own strengths and weaknesses. Some of
these techniques have been combined to detect miRNAs as disease biomarkers in point-of-care (POC)
settings. Nonetheless, there is still potential for further innovation to facilitate the detection of miRNAs.
Key words MiRNA detection, Biomarkers, Point-of-care setting
1 Introduction
RNA molecules are very important regulators of gene expression in

both eukaryotes and prokaryotes [1]. MicroRNAs (abbreviated to
miRNAs) are a class of single-stranded noncoding small RNAs
(sRNAs) around 22 nucleotides in length and ranging from
approximately 18 to 25 nucleotides [2–4]. They were first discov-
ered in the early 1990s [5] but it was not until the early 2000s that
they were first recognized as a specific class of regulator [6–
8]. Although miRNAs were first identified using genetic
approaches, the use of cloning, sequencing, and bioinformatic
approaches has been crucial in the rapid discovery of subsequent
miRNAs [9, 10].
There are differences in miRNA biogenesis between animals
and plants. RNA polymerase II transcribes miRNA genes to form
long primary transcripts (pri-miRNAs) in the nucleus. In animals,
canonical miRNA biogenesis involves a miRNA processing com-
plex consisting of the RNase III endonuclease Drosha and the
cofactor DGCR8 cleaving pri-miRNAs into precursor miRNAs
(pre-miRNAs), which are characterized by a stem-loom structure
[1]. In plants, pri-miRNAs are cleaved to pre-miRNAs
Tamas Dalmay (ed.), MicroRNA Detection and Target Identification: Methods and Protocols,
Methods in Molecular Biology, vol. 2630, https://doi.org/10.1007/978-1-0716-2982-6_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
1
2 Afrah Bawazeer and David C. Prince
predominately by the RNase III endonuclease Dicer-like1 (DCL1),

although examples of other DCLs being involved in miRNA pro-
cessing exist [11]. Pre-miRNAs are then exported from the nucleus
to the cytoplasm by an Exportin-5/Ran-GTP complex (animals) or
Hasty (plants) and then further processed by RNase III endonucle-
ase Dicer (animals). One strand of the duplex is preferentially
loaded into an Argonaute protein to generate a functional
miRNA-induced silencing complex (miRISC), which then facili-
tates gene regulation.
MiRNAs regulate most biological functions in eukaryotes by
regulating gene expression through translational repression or tar-
get degradation [12], although miRNA-mediated translational
activation has also been reported [13]. Therefore, an understand-
ing of miRNAs is necessary to understand the fundamental pro-
cesses of life such as cellular proliferation, differentiation,
morphogenesis, and apoptosis. Changes to miRNA expression
can cause or be an indicator of a wide range of human diseases
including cancer, diabetes, and cardiovascular disease [14]. Analysis
of miRNA expression in specific cell types could hold immense
diagnostic value and the correction of altered miRNA levels in
disease states presents promising therapeutic prospects. MiRNAs
in human body fluids (“circulating miRNAs”) have been shown to
be extremely stable to the action of RNases. Circulating miRNA
levels in serum and plasma appeared to be unaffected by several
treatments including freeze–thaw cycles, long-term storage,
extreme pH, or even boiling [15]. These properties make miRNAs
a promising class of novel biomarkers that could be used for diag-
nosing life-threatening diseases, monitoring of disease progression,
guiding treatment selection, and defining drug dosages. Taken
together, miRNA detection is a key and important concept in
molecular biology from the standpoint of both fundamental
research and medicine.
This review summarizes the detection technologies that exist to
measure miRNAs and describes the advantages and disadvantages
of each of these techniques. As miRNA detection and measurement
is becoming important in the treatment of disease, the use of
detection techniques in the point-of-care (POC) setting is also
briefly discussed.
2 Detection Technologies
2.1 General Detection of miRNAs is challenging because of low abundance,

Challenges in large variability in per cell copy number, short sequence length, and
Detecting miRNAs sequence similarities within miRNA families [4, 9, 16]. Individual
miRNA levels range from a few copies to tens of thousands of
copies per cell. Within a family, miRNAs can differ by a single
nucleotide, and yet each specific miRNA can be differentially
Detection of miRNAs 3
regulated during cellular processes or in disease conditions. Due to

these challenges, some detection methods amplify the signal of the
miRNAs before detection (e.g., RT-qPCR), whereas other meth-
ods detect miRNAs through direct hybridization without amplifi-
cation (e.g., Northern blot).
2.2 Conventional A range of technologies such as Northern blotting, in situ hybridi-

Technologies for the zation, microarrays, RT-qPCR, next-generation sequencing, and
Detection of miRNAs in vivo reporter assays are conventionally used to detect miRNA
in research studies. These techniques will be described below, along
with their associated advantages and disadvantages.
2.2.1 Northern Blot Northern blotting analysis [17, 18] allows information on the size
and abundance of a specific RNA to be determined from a complex
mixture and has been widely used to detect miRNAs since their
initial discovery [5]. Northern blotting to detect miRNAs involves
separating the RNA in a sample by size through denaturing poly-
acrylamide gel electrophoresis. The RNA is then transferred and
cross-linked to a membrane and a nucleic acid probe complemen-
tary to the target RNA is hybridized to the membrane. Probes can
be labelled with radioactivity, fluorescence, or digoxygenin and
detected with an appropriate technique. Efforts have been made
to improve the sensitivity of miRNA detection with Northern
blotting by using locked nucleic acid (LNA) probes and 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated cross-
linking [19–21]. The advantage of Northern blots is that they
simultaneously detect mature miRNAs and miRNA precursors,
therefore providing information on the sequence and length of
miRNA. The disadvantages of Northern blots are that they are
very labor-intensive and time-consuming, require relatively large
amounts of sample, are semiquantitative rather than quantitative,
have low sensitivity to detect miRNAs, and use radiolabelling to
increase sensitivity that adds requirements for special training and
safety measures.
2.2.2 In Situ In situ hybridization involves the use of labelled complementary

Hybridization nucleic acid probes to detect single-stranded RNA (or DNA) in
fixed cells or sections of tissue [22]. LNA probes have higher target
affinity and specificity compared to a traditional DNA oligonucleo-
tide [19]. In situ hybridization allows miRNA expression and local-
ization within a cell or tissue to be visualized. The use of
fluorescently labelled in situ hybridization probes (miRNA FISH)
allows for multiple miRNAs to be visualized together [23, 24]. The
advantage of in situ hybridizations is that information is provided
on the spatiotemporal expression of the miRNA. The disadvantages
of in situ hybridizations are that they are low throughput and not
typically quantitative.
2.2.3 Microarray Microarrays are widely used for gene expression analysis studies and
allow parallel analysis of already described miRNAs [25]. The cen-
tral concept relies on hybridizing labelled miRNAs in a sample with
a probe specific to a miRNA in order to measure the abundance of
specific miRNAs through fluorescence-based detection. Hundreds
of different probes are usually bound on a solid surface such as a
glass plate to form the microarray. Sample RNA is reverse tran-
scribed to cDNA and labelled with fluorophores or biotin, to which
a streptavidin-labelled fluorophore can be added later. The labelled
cDNA is hybridized to the microarray, and unbound cDNA
removed by washing and fluorescence quantified. The advantages
of microarrays are that a large number of miRNAs can be quantified
simultaneously in a single experiment and the amount of RNA
required is low. The disadvantages of microarrays are that only the
miRNAs on the arrays can be quantified, miRNAs with high
sequence similarity can cause specificity to be compromised, special
expertise is required for data analysis, and data often require experi-
mental validation with an additional method (such as RT-qPCR).
2.2.4 RT-qPCR Quantitative reverse transcriptase polymerase chain reaction

(RT-qPCR) is regarded as the gold standard approach to quantify
miRNAs as it provides a good balance between cost, precision, and
sample size along with a large functional dynamic range [26]. The
extracted RNA is converted into cDNA by reverse transcription
using universal, miRNA-specific, or miRNA-specific stem–loop
primers. The presence of the mature miRNA sequence in
pre-miRNAs and pri-miRNAs can cause challenges for specific
and sensitive primer design; however, stem–loop primers can dis-
tinguish between different miRNA forms [27], enhancing specific-
ity and reproducibility [28]. The cDNA is then used for qPCR
either with a miRNA-specific forward primer, a universal reverse
primer and SYBR Green, or TaqMan probes. The advantage of
RT-qPCR is that it can be designed to be highly specific and
quantitative. The disadvantages of RT-qPCR are that the process
is time-consuming, has low throughput, and requires expensive
thermal cycling equipment, and designing a highly specific and
quantitative assay requires expertise and the optimization of several
different steps.
2.2.5 Next-Generation Next-generation sequencing (NGS) refers to sequencing technol-

Sequencing ogies such as Illumina sequencing, which were developed after
Sanger sequencing (first-generation sequencing), and can be used
for various lengths of RNA and DNA [29]. Profiling miRNAs with
NGS involves ligating 3′ and 5′ adaptors to the isolated miRNA,
generating cDNA through reverse transcription, amplifying the
cDNA by PCR, and then sequencing with an appropriate technol-
ogy. Multiple samples can be sequenced in the same run and
subsequently separated computationally. The advantages of NGS
are that it is high throughput, can detect all miRNAs present

(including previously unknown miRNAs), and avoids the back-
ground noise and cross-hybridization problems of microarrays.
The disadvantages of NGS are that it is expensive, library prepara-
tion and sequencing can lead to PCR biasing issues, it requires
computational infrastructure and expertise for analysis of the data,
and it requires further experimental validation to distinguish
between meaningful data and noise or false positives.
2.2.6 In Vivo The levels of specific miRNAs can be measured in time and space by
Reporter Assay using in vivo reporter assays. By fusing a fluorescent protein to a 3′
UTR with a specific miRNA binding site, the level of a specific
miRNA can be determined by detecting fluorescence, with an
inverse relationship between fluorescence and miRNA level. For
example, levels of miRNA let-7a were measured by fusing GFP to
the 3′UTR of Ras, which encodes a let-7a binding site [30]. The
advantage of in vivo reporter assays is that miRNA activity levels can
be determined in time and space within living cells. This important
information is lost when using methods that require RNA extrac-
tions from tissues. The disadvantages of in vivo reporter assays are
that they can often be low throughput and time consuming, they
require prior knowledge of a miRNA’s target site, and they require
the ability to genetically manipulate the organism being studied.
2.3 Emerging Conventional methods for detecting miRNAs are widely used for
Technologies for the research purposes; however, their limitations make them less suit-
Detection of miRNAs able for clinical uses such as point-of-care (POC) diagnostics. A
range of miRNA detection technologies have emerged over recent
years that offer advantages for detecting miRNAs for research
purposes but also have the potential to be more suitable for POC
testing. Oligonucleotide-templated reactions do not amplify the
miRNA signal but need to be coupled with a detection method.
Enzyme-free oligonucleotide and enzyme-based signal amplifica-
tion techniques increase the signal of the miRNA in the assay but
need additional methods to detect this signal. Nanostructured
materials can be used to amplify or detect miRNA signals. Electro-
chemical and capillary electrophoresis methods can be used to
detect the signal generated by other techniques. These techniques
will be described below, along with their associated advantages and
disadvantages.
2.3.1 Oligonucleotide- Oligonucleotide-templated reactions (OTRs) [31] involve the

Templated Reaction sequence-specific Watson–Crick base pairing of two nucleic acid
probes to a target sequence, in this instance a miRNA. The probes
are designed with probe heads that generate a measurable signal
when they react. Binding to the target increases the effective molar-
ity of the two probes, catalyzing a highly unfavorable chemical
reaction from which the signal is then detected. OTRs have been
engineered to produce optical or electrochemical readouts

[32]. The advantages of oligonucleotide-templated reactions are
that they can be engineered to be highly specific, isothermal, and
amplification-free, have very low background signal, and be highly
cost-effective [33]. The disadvantage of oligonucleotide-templated
reactions is that reaction conditions need to be compatible with
oligonucleotide hybridization, although peptide nucleic acids or
LNA can be used as alternatives.
2.3.2 Enzyme-Free Hybridization chain reaction (HCR) [34] and catalytic hairpin
Oligonucleotide Signal assembly (CHA) [35] are isothermal, enzyme-free amplification
Amplification techniques that use toehold-mediated DNA strand displacement
(TMSD). Both techniques involve two different DNA hairpins; a
miRNA (the “toehold”) hybridizes to the first DNA hairpin, which
then initiates binding to the second DNA hairpin to form a DNA
duplex. In HCR the duplex then sequentially hybridizes with fur-
ther hairpins in a chain of reactions to assemble a long, nicked
double-stranded DNA, while in CHA the duplex between the hair-
pins releases the miRNA to catalyze further assemblies. The
miRNA-mediated double-stranded DNA products of HCR and
CHA can then be detected with a range of methods including
fluorescence, electrochemistry, colorimetry, and chemilumines-
cence. The advantages of enzyme-free oligonucleotide signal ampli-
fication techniques are that they are simple, fast, cost-effective,
sensitive, and specific, and the lack of enzymes remove the need
to precisely control temperature, pH, and buffer conditions. The
main disadvantage of enzyme-free oligonucleotide signal amplifica-
tion techniques is that designing the DNA hairpins required can be
complex.
2.3.3 Enzyme-Based A range of isothermal enzyme-based signal amplification methods

Signal Amplification have been used and developed for miRNA detection. Rolling circle
amplification (RCA) [36] and its various derivatives (reviewed by
[37]) and loop-mediated isothermal amplification (LAMP)
[38, 39] use polymerases, and exponential amplification reaction
(EXPAR) [40] and strand-displacement amplification (SDA) [41]
combine polymerases with nucleases, while other methods such as
duplex-specific nuclease signal amplification (DSNSA) [42] only
feature nucleases. The products of enzyme-mediated signal ampli-
fication can then be detected with a wide range of methods includ-
ing Northern blotting, fluorescence, electrochemistry, colorimetry,
and nanoparticles. The advantages shared by enzyme-based signal
amplification methods are that they are isothermal and therefore do
not require thermal cycling equipment and they can be highly
sensitive and specific. The main disadvantage with many enzyme-
based signal amplification methods is that probe design can be
complicated.
2.3.4 Nanostructured Nanostructured materials are a broad term that includes nanopar-
Material-Based ticles and other types of nanomaterial. A variety of nanostructured
Amplification and Detection materials including gold nanoparticles, silver nanoparticles, carbon
nanomaterials, and quantum dots have been used in miRNA signal
amplification and miRNA detection (reviewed by [43–45]). The
versatile nature of nanostructured materials means that they are
often used in conjunction with other signal amplification (e.g.,
enzyme-based) or detection (e.g., electrochemical) techniques.
The advantages of nanostructured materials are high sensitivity,
stability, biocompatibility towards nucleic acid probes, and good
optical properties. The disadvantage of nanostructured materials is
that a specialized nanotechnology platform is needed.
2.3.5 Electrochemical Electrochemical detection measures an electrical signal produced

Detection by a molecular recognition event. The molecular recognition of
miRNAs is achieved by hybridization to a complementary nucleic
acid probe. Electrochemical detection of miRNAs is mainly accom-
plished through amperometric sensors [16], which rely on the
oxidation or reduction of a molecule to produce an electrical signal.
The advantages of electrochemical detection are specificity, simplic-
ity, portability, ability to be miniaturized, cost-effectiveness, rapid
output, and broad dynamic range. The disadvantage of electro-
chemical detection is poor sensitivity for lowly expressed miRNAs.
However, this can be greatly increased by amplifying the signal of
such miRNAs using nanoparticles or isothermal amplification.
2.3.6 Capillary- Capillary electrophoresis [46, 47] is a method to separate biomo-

Electrophoresis- lecules based on their electrophoretic mobility when a voltage is
Based Detection applied. The technique has been applied in various ways to detect
miRNAs (reviewed by [48]) with capillary-electrophoresis-based
hybridization assays showing particular promise in the sensitive
detection of multiple miRNAs [49–51]. The advantages of
capillary-electrophoresis-based detection are that the probe design
is simple and multiple targets can be detected at once. The disad-
vantage of capillary-electrophoresis-based detection is that some
applications require specialized equipment.
2.4 Point-of-Care Detection of miRNA in a POC setting to aid clinical decisions

(POC) Detection of needs to be convenient, robust, rapid, and cost-effective. The
miRNAs limitations of conventional methods for detecting miRNAs mean
they are rarely suitable for POC detection. However, emerging
methods for detecting miRNA are being combined to engineer
devices that are more suitable for POC detection. The most
promising examples are lateral flow and microfluidic devices and
will be described below, along with their associated advantages and
disadvantages.
2.4.1 Lateral Flow Assay Lateral flow assay (LFA) devices are familiar to many for the testing
Devices of hormones associated with pregnancy or viruses such as corona-
virus [52]. A wide variety of labels including antibodies, nanopar-
ticles, or OTRs can be used to output a colorimetric or
fluorescence-based reading. Several LFAs have been developed to
detect miRNAs; for example, an OTR-based LFA was developed to
detect miR-150-5p [53], and a gold nanoparticle LFA was devel-
oped to simultaneously detect miR-21, miR-155, and miR-210
[54]. There are many advantages to LFAs: the versatile format
allows many different types of recognition molecules, labels, and
detection systems to be used, they can be manufactured cheaply
and in large quantities, they often require no specialist equipment
or training to use, they can be stable in a wide range of environ-
mental conditions and settings, results are often available quickly,
and they can be very sensitive and specific. The principal disadvan-
tage of LFAs is that many produce qualitative or semiquantitative
results, although efforts are currently being made to improve
sensitivity [55].
2.4.2 Microfluidic Microfluidic devices process or manipulate small volumes of fluids

Devices using channels with small dimensions [56]. In the context of
detecting miRNAs, microfluidic devices combine precisely con-
trolled small volumes of liquid and the emerging signal amplifica-
tion and detection techniques outlined above to determine levels of
specific miRNAs in a biological sample. Several microfluidic devices
have been developed to detect miRNAs; for example, a microflui-
dics platform with a complementary molecular beacon probe has
been developed to detect miR-21 in the serum of breast cancer
patients [57], and techniques including EXPAR and quantum dots
were integrated onto a microfluidics device to simultaneously ana-
lyze miR-21 and miR-155 from tumor cell lysate [58]. The advan-
tages of microfluidic devices are simplicity, sensitivity, rapidity, cost-
effectiveness, portability, and the potential to integrate all steps
(such as sample processing and miRNA detection) onto a single
device. The disadvantage of microfluidic devices is that they can be
complicated to design.
3 Conclusion
The detection of miRNAs is necessary for both a greater under-

standing of their functions in biology and their use as biomarkers in
disease treatment. However, inherent properties of miRNA make
their detection challenging. All miRNA detection methods have
strengths and limitations, with conventional laboratory-based
methods necessary to discover and characterize miRNAs in detail,
while emerging methods can be combined to simplify the detection
of characterized miRNAs. Progress has been made in deploying
miRNA detection techniques to the POC setting for disease treat-

ment but there is much potential for this to be further developed
and optimized in the future.
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CHAPTER V.
EXPELLED.
The next morning, leaving Madeline at the station to follow by a

later train, Mr. Wynne called at Harperton, in order to have a little
explanation. The maid’s face (she was an old maid) looked
portentously solemn as she opened the door; and—oh! ominous
objects!—two good-sized basket trunks, and a bonnet-box, stood
waiting in the hall. As he glanced at them in passing, some one
came out through a door just behind him, and said, in a biting tone—
“Dear me! I am surprised to see Mr. Wynne under the
circumstances; but, as he is here, perhaps he can give an address
for Miss West’s boxes?”
“May I ask what you mean, Miss Selina?” he said, turning to
confront her the instant the drawing-room door was closed.
“I mean,” she replied, flushing to a dull brick colour, “that after her
escapade of last evening, Miss West never enters this house again
—a young lady who stayed out all night!” she concluded with a wild,
dramatic gesture.
“But, you know, that was not her fault, Miss Selina. We waited
exactly where you told us—at the bottom of the steps—and so
missed the train. I could not get a cab, though I did my utmost, the
snow was too deep. I left Miss West at the Railway Hotel and
brought her from there this morning. She——”
“Oh,” interrupted his listener, throwing up both hands, “pray spare
me the details! It is nothing to me whom she was with, or where she
went. We have quite done with her. It was a planned thing between
you, no doubt.”
“Miss Selina,” cried Mr. Wynne, “your sex protects you! A man
dared not say what you have permitted yourself to utter, and do not
in your own heart believe. Am I to understand that because, through
waiting for you, by your own express direction, Miss West lost her
only train home last night, and was obliged to remain in Riverside,
you would blast her reputation, and thrust her out of doors?”
“You are!” she returned, defiantly, looking him full in the face with
her cold, cruel, little eyes.
“And may I ask what is to become of the young lady?” he inquired,
with a forced calmness that was ominous enough.
“Nay,” shrugging her shoulders, “that is a matter between her and
you.” Then she added, with an evil smile, “She need not refer to us
for a character.”
“Perhaps your mother will be more lenient,” he said, making a
great effort to restrain his temper. “Remember that Miss West has no
home and no friends. Can I see Mrs. Harper?”
“I am speaking for my mother,” she answered sharply. “She
refuses to see the girl, or allow her inside our door. There is no use
in your persisting—it is waste of time. We are not rich, but, at any
rate,” choking with excitement, “we have always been respectable!”
“I am delighted to hear it,” he replied, making a low, ironical bow;
“and as there is nothing further to be said, I will wish you good
morning.”
“Good morning!” replied Miss Selina, ringing the bell, and
curtseying simultaneously. “You will be pleased to remove Miss
West’s boxes at once, and inform her that letters from her will be
returned unopened”—thereby securing the last shot, and the last
word. And Mr. Wynne walked out of the house in a bewildered and
confused state of mind, outwardly cool, but in reality at boiling point.
He had not proceeded far when he met Madeline coming towards
him, with a terrified and expectant face. Now was the moment for
action. His senses were stung to alertness, his mind cleared of
misgivings; he made a desperate resolve. She was thrust out
homeless and alone in the wide, wide world! She should share his
home, such as it was; it was better than none. She should, an she
would, be his wife—and rich in love if in nothing else. Prudence had
hitherto sealed his lips—for her sake chiefly. Now that she had no
resources, no place open to receive her, he could and would speak.
The first thing he did was to hail a cab, and despatch the man
straight back to Harperton for Miss West’s luggage, desiring him to
bring it to the station.
“Why, what does it mean? Are they so very angry?” she asked
with blanched cheeks. “Oh, you don’t mean that they are sending me
away?” For she noticed that Mr. Wynne looked unusually pale and
grave.
“Come down here with me,” leading her into some public gardens
that they were passing, “and I will tell you all about it.”
The gardens were miserably wintry. Snow lay on the ground, a
couple of boys were snowballing, some starving red-wings fluttered
across the path, a granite-grey sky lowered overhead. Surely it was
the last place on God’s earth in which to relate a love tale; and the
girl herself, what a picture of misery! Oh! thought the young man, if
Mrs. Wolferton had but been at home—but, alas! she was abroad—
she would have been a true friend to this poor forlorn child. Madeline
was, of course, wearing her evening dress, such as it was—at any
rate, it was thin. A shabby little plush opera cloak barely covered her
perishing neck and arms. Over this was drawn a meagre black cape.
On her head she wore a sunburnt sailor hat; in her frozen, mittened
hand she held a fan; her face was pinched with cold, and white with
anxiety. No lovely lady-fair was here to woo this bleak January
forenoon. And what of ambition—the stern, jealous mistress to whom
he was pledged?
“They are very angry, senselessly angry,” began the young man.
“They won’t take you back again, and have actually packed your
boxes ready for removal. However, when one door shuts, another
opens. There is a home ready for you, Madeline. Can you guess
where it is?”
She gazed at Mr. Wynne, and stood perfectly still and very white,
with her thin, sensitive lips tightly pressed together, and made no
reply.
“You know that it is my home,” he continued eagerly. “I need not
tell you that I love you, and so well do I love you, that until now I
have never dared even to whisper my love. I am poor, I have my way
to make as yet, it may be a life of struggling poverty. Can you share
it—will you venture, Madeline?”
The girl stepped back a pace, and suddenly sat down upon an iron
garden bench, still silent, and covered her face with her mittened
hands.
“Will you not answer me!” he pleaded. He dared not remove her
hands, or offer her a caress. The snowballing had ceased; the
present scene in real life attracted the two boys, who had drawn
near. The lady was sick, or looked like it.
“You do not mean it,” she faltered. “I know you are very, very kind,
but I cannot accept your pity, for that is what it comes to.”
“I solemnly declare to you that it is not,” he rejoined with emphasis;
“but even if it were, have you not heard that pity is akin to love?”
“It is utterly impossible,” she said slowly. “You are speaking out of
the goodness of your heart, on the impulse of the moment. This time
yesterday, tell me honestly,” raising her lovely eyes to his, “had you
any intention of—of—of this?”
“To be truthful, then, I had not.”
“There, you see, that is enough. There is your answer,” with a
quick little gesture.
“No, no, hear me out. It was on your account that I held my
tongue. If I had had a decent income I would have spoken to you
long ago; but I felt that I had no right to remove you from Mrs.
Harper’s care without having a comfortable home to offer you. I
meant to work very hard and to return next year. Now all has been
changed. Circumstances alter cases. I ask you now, Madeline, will
you be afraid to begin with me at the bottom of the ladder—
something tells me that I shall reach the top?”
“I shall only be a dead weight and a burden,” she replied in a
broken voice. She was relenting. Her own heart was an eloquent
advocate for Mr. Wynne.
“What will your relations say when they hear that you wish to
marry a portionless girl, a—beggar?” she murmured tremulously.
“They will say nothing that can affect us. I am independent. I have
no claims on them, and they have no right to dictate to me. By the
time they hear the news, we shall, I hope, be married. We have
nothing to wait for, and the sooner you have a home of your own the
better. I wish I had a sister or some near relative that I could take you
to, but I am almost as much alone in the world as you are.”
In the end Mr. Wynne prevailed—was not talking his trade?—and
Madeline West walked out of that wintry white garden his affianced
wife.
Rash young man! Rash young woman! One would have thought
that they had the wealth of Crœsus, the full consent and warmest
wishes of tribes of wealthy relations, to look at their faces as they
passed through the gates side by side.
Miss West did not feel the snow soaking through her thin walking
shoes. No, she was treading on air—had thrown all doubts and
misgivings to the winds, and was prepared to make the most of this
heaven-sent period. She was about to enter on a new and happy life,
believing that, although a poor man’s wife, her path would be strewn
with roses.
She had about as much practical experience of household cares—
the value of pounds, shillings, and pence—as one of the children in
the third class at Harperton. As for Laurence Wynne, Madeline was
his, Madeline was an angel, young, unspoiled, and unsophisticated,
with modest wishes, and a firm faith in him. Their future was before
them! It was!
CHAPTER VI.
“POVERTY COMES IN AT THE DOOR.”
In a very short time Madeline West was Madeline Wynne. She

was married at a little old church in the City, with no other witnesses
than the verger and the clerk; and Mr. and Mrs. Wynne spent a week
in Paris ere they set up housekeeping, in modest lodgings not far
from the Temple, and from which, by leaning well out of the drawing-
room window, and nearly dislocating your neck, you could obtain a
glimpse of the Thames Embankment.
The good old days, when Traddles and Sophy lived in chambers,
and entertained half a dozen of “the dear girls,” were no more. Mr.
Wynne was obliged to set up his little tent outside the venerable
precincts, in the second floor front of Solferino Place. To Madeline it
was a palace, because it was her very own. Here she might poke the
fire, alter the arrangement of the furniture, pile on coals, order tea at
any time, and go out and come in as she pleased. She could
scarcely realize such liberty! Neither could she realize her wedding-
ring, and she frequently stared for a moment in doubt when she
heard herself called “Mrs. Wynne.”
Laurence was not so poor as she imagined, for he hired a piano,
bought her songs, flowers, and—oh! joy—three such pretty new
dresses; he took her to the theatres, for walks in the parks (when he
had time), he showed her most of the sights of London—St. Paul’s,
Westminster Abbey, the National Gallery, and the Tower.
He was even extravagant in one line. He laid out for her a reckless
amount of shillings and half-crowns on literary papers, magazines,
and books. Laurence was fond of reading; she was not, and she little
knew how she startled him when she exclaimed, “Besides all the
other hateful things you have delivered me from, Laurence, you have
delivered me from books! I never wish to open one again!”
Now Laurence had been looking forward to introducing his pretty
Madeline to all the great masters in English literature, to hearing her
fresh comments, to sharing her raptures, to comparing first
impressions, favourite pieces, favourite characters; in short, to
opening for this girl of eighteen the portals of a new world. Alas! it
soon became evident that Madeline had an absolute lack of literary
taste. She had a taste for music, for flowers; a marvellous taste in
colours, and in dress; but for reading, as he understood it, not an
atom. (At first he had had visions of reading her some sketches and
articles of his own, but soon changed his mind, and kept his MS. in
his writing-desk.) He read aloud well, and selected, as he believed,
gems; but, unfortunately, Mrs. Wynne preferred paste!
Lamb’s essays were “quite too awfully dry.” Wordsworth was ten
times worse—she could hardly stifle her yawns. And even when he
was reading “Silas Marner,” and, as he considered, George Eliot’s
masterpiece, he noticed that Madeline was shyly perusing the
advertisements in a ladies’ newspaper. She looked so nonplussed
and unhappy if he paused and suddenly asked her, “If that was not
fine? and how such and such a passage struck her?”
At length he relinquished his efforts. It was time, when Madeline,
with a pretty pout, said, “My dear Laurence, I might as well be at
school; you are just talking like Mr. Falk, our professor of English
literature. Such an ugly little mummy.”
“And to whom you never listened?”
“Not I; and I never could remember names, periods, or dates. You
must make the best of me. In some ways you will find that I am
hopelessly stupid.”
In spite of these tiresome readings, Madeline was thoroughly
happy; there was not one single drawback, not one little cloud on her
sky, if we except an occasionally heavy magazine article to which
she was obliged to lend her ears. And Laurence was happy too. It
was delightful to come home those dark, wet nights, and find a kiss,
a blazing fire, and his pretty Madeline awaiting him. She was always
smiling, always so ready to see the comic side of everything, a
veritable sunbeam in that drawing-room.
“Who would be a bachelor?” he asked himself contemptuously, as
he watched her flitting to and fro after dinner, pulling up his armchair
and filling his pipe. If he had one little arrière pensée, it was this, that
she would not always give him mutton chops, and a wish that her
ideas of a menu were a little more expansive.
Nevertheless he was perfectly content. He had an incentive to
work hard now, and he did work. He was getting known in a small
way. He had the gift of oratory, of what is known as legal tact, a
handsome presence, and the power—given to so few—of swaying
men’s minds with his eloquence, as the flame of a candle in the
wind. But, then, he was only twenty-eight—a mere boy in the eyes of
the ancient profession, where a man begins to make a start about
fifty. Still Laurence Wynne had his foot on the lower rung of the
ladder. More than one shrewd solicitor had noted him. His luck had
turned; his marriage had brought him good fortune, though it had
scared away all his relations, and he had completely dropped out of
society.
But this fool’s Paradise was not to last—it never does. The angel
that opened the gate, and drove the foolish pair out into the
everyday, hard, stony world was typhoid fever.
The hot summer succeeding their marriage was a trying one, and
in the sultry September days typhoid fever laid hold on many victims,
among others on the hard-working young barrister—seized him with
a death-like grip, flung him on a sick bed, and kept him there for
months.
The fever was so difficult to shake off, and it had brought so many
other ills in its train. Finances were low—as they are sure to be when
the bread-winner is idle. Doctors’ bills and chemists’ bills were
mounting up, as well as the butcher’s and baker’s, not to speak of
the landlady’s little account.
All the burden now lay upon one pair of young shoulders—
Madeline’s; and, to quote a homely but expressive phrase, she
absolutely did not know where to turn. She had neither money nor
friends. Her husband had no capital; his slender fortune had been
invested in his education and profession. And as to his friends and
his distant connections, they had disowned him. When they had
heard of what they were good enough to call “his low marriage with a
teacher in a school,” they had washed their hands of him with
wonderful unanimity. Society had lost sight of him for months; Mr.
and Mrs. Wynne had no acquaintances. Poor Madeline was in
terrible straits, but her courage rose with the occasion; she was
brave and energetic, and did not sit down with her hands before her
and cry.
A schoolfellow of her husband’s (another young barrister) came to
see her and him, and gave help in the shape of advice, which for
once was valuable. They moved to the top story—the attics. (That
was a step of which their landlady highly approved.) And he
procured some law copying for Madeline—who wrote a clear, neat
hand—which brought in a few shillings, and kept the actual wolf from
the door. He sent fish, grapes, and other little delicacies to the
invalid, and was in truth that rara avis—a friend in need.
He considered that Wynne had behaved like a madman in
marrying on nothing; but certainly the girl was an immense
temptation—so young, so pretty—such eyes he had never seen—so
unsophisticated and fresh, and yet possessing excellent sense and
an elastic and dauntless spirit. Here for once was an instance in
which poverty had not thrust love out of the window. Strange, but
true, their reverses had only served to draw the Wynnes more
closely together. They afforded a refreshing study to Mr. Jessop, who
was a cynic and a philosopher in a small way, and who sneered and
snarled and marvelled. Things had not even come to the worst with
these unfortunate people, not until a third was added to the
establishment in the shape of a Master Wynne, who puckered up his
wrinkled red face, thrust his creasy fists into his eyes, and made
hideous grimaces at the world in which he found himself—and in
which, to tell the truth, he was not particularly wanted, except by his
mother, to whom he was not only welcome, but, in her partial eyes, a
little household god!
His father, who was slowly recovering—an emaciated spectre of
what he had been—was dubious with regard to the striking
resemblance to himself, and frequently wondered in his inmost soul,
as to what was to be the future of his son and heir? How was he to
be fed, clothed, and educated? Dismal echoes answered, “How?” for
the Wynnes were now desperately poor.
I mean by this, that Mr. Wynne’s watch had long been ticketed in a
pawnbroker’s window, that Madeline’s one little brooch had gone the
same way; also—oh, breathe it not!—her best gown and hat; also
Mr. Wynne’s top coat and evening dress clothes; that the invalid
alone tasted meat—and in scanty portions—Madeline telling many
clever fibs with regard to her own dinner. Her inexhaustible spirits
and vivacity seemed to sustain her—that, and a little bread and tea.
The one person who was well-to-do was the baby. He was clothed
in a beautiful cloak and hood—Mr. Jessop’s gifts—purchased, with
many blushes, by that keen-eyed, close-shaven gentleman, and
presented with pride to his godson and namesake. More than once
Madeline’s mental eye had seen these sumptuous garments
smuggled away to the pawnbroker’s round the corner, but she fought
hard with the idea, and had sternly kept it at bay—as yet. Their
circumstances were, indeed, all but desperate, when one evening
Mr. Jessop came thundering up the stairs, newspaper in hand, and
panted out, as he threw himself into the nearest chair and took off
his hat—
“I say, Mrs. Wynne, what was your name before you were
married?”
“My name,” she echoed, looking blankly at him, for she was trying
to keep the baby quiet and to do some copying simultaneously—vain
and exasperating task—“was West—Madeline West.”
“Ah! I thought so!” he cried triumphantly, clearing his throat and
unfolding his paper with a flourish.
“Then just listen to this:—‘Madeline West.—If this should meet
the eye of Madeline Sidney West, she is earnestly implored to
communicate with Mrs. H. of H. House, at once, when she will hear
of something greatly to her advantage.’ Now what do you think of
that?” he demanded of his friend, who, drawn up near a handful of
cinders, had been poring over a law book. “Looks like a legacy,
doesn’t it?”
“Too good to be true, I’m afraid. Eh, Madeline?”
Madeline turned her face alternately on the two men. A faint colour
had invaded her thin, white cheeks, and her eyes brightened as she
said—
“There is no harm in answering the notice; it may mean
something.”
“Why, of course it does,” cried Mr. Jessop, emphatically. “Get a
pen, give me the infant, and write a line now, and I’ll post it.”
And Madeline accordingly sat down and wrote to Mrs. Harper on
the spot, whilst her companions watched her in silence.
“Dear Mrs. Harper,
“I have seen your notice in the Times of to-day. My address
is—2, Solferino Place, Westminster.
“Yours truly,
“M. W.”
She was so accustomed to sign merely her initials, and was so
flurried between anticipation, anxiety, excitement, and the screams
of the baby, that she never had the presence of mind to write her full
name, and on this slight omission, this one little cog, turned a most
important factor in her future career.
CHAPTER VII.
A TELEGRAM FOR MISS WEST.
The very morning after Madeline had despatched her letter, a

telegram was handed in for Miss West, 2, Solferino Place. The
landlady herself mounted, breathless, to the attics, with the tan-
coloured envelope in her hand.
“I was just for sending it away, Mrs. Wynne,” she gasped,
surveying her with an inquiring eye; “but it came into my mind as I’d
show it to you on the chance.”
“Thank you; it is for me,” rejoined her lodger, hastily tearing it open
and running her eyes over it. As she read, she became crimson with
amazement and agitation. “Come at once—to-day if possible. News
of your father.—From Mrs. Harper, Streambridge,” was the message.
“But it’s for Miss West, and you’ve gone and opened it!” exclaimed
the landlady, suspiciously. “How is that, eh? I never would have
supposed—no, never,” folding her arms belligerently, “as you wasn’t
on the square; and as I’ve allus kep’ a respectable ’ouse, I couldn’t
think——”
“You need not think, Mrs. Kane; you need not alarm yourself about
the matter, it is all quite right. I am Mrs. Wynne, but I was Miss West
once upon a time. The sender of the message does not know that I
am married,” interrupted Madeline, speaking with studied composure
—though her heart was beating fearfully fast.
Insolent as Mrs. Kane was, she dared not quarrel with her. Her
roof covered them on sufferance. Were she to thrust them forth,
where were they to go? They were entirely at her mercy, for they
owed her money; and latterly she had been inclined to take out a
large amount of interest in rude insolence, biting gibes, and
unpleasant hints with regard to “paupers a coming and settling down
on poor, honest, hard-working people—paupers as could afford
dress, and theatres, and pianos once, and saved nothing for a rainy
day!”
Paupers—impecunious people like the Wynnes, especially Mrs.
Wynne, who bore the brunt of these encounters—could not afford to
stand on their dignity, and be independent and “move on.” They must
submit humbly; but it was insufferably galling—as galling to Madeline
as Miss Selina’s yoke, that had pressed upon her so sorely but one
little year ago.
Who but herself knew with what deprecating eyes and voice she
had pleaded with her impatient landlady for a little time, how humbly
she ventured to ask for coals, how stealthily she crept up and down
stairs, carrying baby, and doing her own miserable errands, making
her presence as unobtrusive as possible, for fear of offending her
hostess’s threatening eyes.
The hostess’s threatening eyes were fixed upon her now, with a
look that was an insult, as she listened to her hurried explanation
with a down-drawn lip.
“Oh, well, I suppose, as I know no better, I must believe you,” and
with a noisy sniff that intimated quite the reverse, Mrs. Kane glared
once round their squalid sitting-room, to see if anything were broken
or missing, or the valuable property damaged in any way; and, failing
to discover the smallest pretext for complaint, passed out of the
apartment with a heavy and aggressive strut, and banged the door
behind her.
Madeline lost not a second in rushing to the invalid with the great
news, and placing the slip of pink paper in his hand.
“There is something at last! I feel that a change is coming; these
terrible days cannot—cannot go on for ever. I believe my father is
alive, and coming home! What do you think, Laurence?” she asked,
and her voice trembled.
Laurence, still holding the telegram in his thin, transparent hand,
gazed at his wife for some seconds in silence. How changed she
was, he thought, with a pang of self-reproach. She was shabby—
very genteelly shabby. Her black dress was all mended and pieced,
her face was haggard, her eyes sunken, their look eager, anxious,
almost desperate.
An intelligent spectator would have declared that she was
obviously half-starved, and so she was. But how furiously she would
have disclaimed such a pronouncement. She would rather have died
than have admitted that truth. As long as Laurence had meat once a
day, as long as baby had milk, she did very well on anything, and
anything may mean almost nothing—it is an elastic word.
Meanwhile, Laurence had been telling himself that he had been a
culpable wretch to marry Madeline West. What would he say to her
father when he placed his daughter in his arms—a daughter in all but
rags, with a face pinched with famine, without a friend, without a
penny, and weighted with a dying husband and a peculiarly ill-
tempered baby?
How much better would it have been if he had curbed his foolish
fancy—nipped it in the bud, and left Madeline to her fate. Why had
he not wired to Mrs. Wolferton? What would her father say? Would
he cast her off?
Madeline had hinted that, as well as she could judge her father
from his letters, he was fond of show and style and great people. He
wished her to dance and sing and play well, and to speak French;
but he had never said a word about literature, or the English
classics, or what Laurence called “the higher education of women.”
On the other hand, he hoped that she would always make
acquaintance with girls her equals, or even superiors, and never
lower herself by school-friendships that it would be impossible for
him to recognize. Madeline had once innocently repeated this to her
husband verbatim, and it came vividly before him now. Madeline had
done more than form a friendship of which her aspiring parent would
disapprove, a friendship that could be slipped out of like an old
glove. Here she was tied for life to a poor man, whose only career
seemed likely to be that of an invalid—a stone round her neck as
long as he lived.
He had but faint hopes of his own recovery; everything was
against it. He knew that this could not be helped, and he was very
patient. If he had good wine, wholesome delicacies to tempt his
appetite, instead of gruesome scraps of stale, ill-cooked meat and
poisonous port at a shilling; if he could have a change to pure,
invigorating air, he might yet have a chance. And he knew that he
might as well long for the moon—for the entire firmament!
“What is to be done, Laurence?” asked Madeline, rather surprised
at his long silence. “What do you think of it?”
“You must go, of course,” he returned at last. “Go to-day.”
“To-day! My dear Laurence, what are you thinking of?” sitting down
on a rush chair as she spoke, and staring at him in amazement.
“Where is the money to come from? Look here,” producing a shabby
little purse, with a brass clasp, and turning out the pitiably small
contents, “all I possess is two and sevenpence.”
“Still you must go, Maddie, by hook or crook; much may depend
on it. A return third-class——”
“A return third-class would be twenty-two shillings—one pound
two,” she interrupted. “And, besides, I could not go in this,” looking
down at her gown; “now,” appealingly, “could I?”
“No, you could not,” he replied, with a little flush on his pale face.
“And you must get something out. To get something out something
else must go in. And,” speaking with an effort, “I never thought to
part with them, but they could not go in a better cause. I mean,”
wiping his damp forehead, “my mother’s miniature and my father’s
medals. The miniature is framed in seed pearls; the back is gold—it
ought to fetch a couple of pounds. It’s in my desk, Maddie, in a little
morocco case.” There were other things in his desk—neatly-copied-
out manuscripts. These, alas! were valueless—he had proved them.
“Take it, my dear, and welcome; and the medals—they will fetch a
few shillings.”
“Oh, Laurence,” suddenly kneeling down beside him, “I don’t like
to! Must I really? I know you think so much of them. They are the
only relics you possess. No, no; I really can’t!”
“Yes, you can, and you shall,” said the sick man, with sudden
decision. “Here, at last, is an opening for you, my poor Maddie.
Something tells me that your father is alive—is coming home rich.
You are his only child, his heiress. You will be looked after and
cherished when I am gone. Yes, my dear, it will be best for you in the
end. It was most wicked of me to marry you. I see it all now, only too
plainly. I had put by nothing for such a strait, and I had no wealthy
friends. But I never dreamt that it would come to this, Maddie;
believe me, I never did. Forgive me!” he urged, and tears, born of
weakness and remorse, stood in his hollow eyes.
“Laurence!” she interrupted, attempting to place her hand on his
mouth.
“I should have walked home in the snow that night; I should have
taken you to the Wolfertons’ house, and telegraphed for her; I should
have gone to the parish clergyman—done anything but what I did,
and which led to my dragging you into such a pit as this!” with an
inclusive wave of his hand and a glance round the mean little attic.
“But it won’t be for long now,” he added in a lower voice.
“Oh, Laurence,” she almost screamed as she seized his arm, “why
are you telling me such terrible things, when we have a little gleam of
hope at last? It is cruel—cruel of you. You couldn’t mean that, after
all we have gone through together—that when we are approaching
smooth water—you—you would leave me!”
And here she suddenly broke down and burst into tears, for, alas!
she had an agonizing inward conviction that there was truth in what
he said. How pale and thin and wasted he looked! No one would
recognize him who had seen him last year, with his shorn head,
gaunt cheekbones, and sunken eyes; and she had a heart-breaking
feeling that it was not mere actual illness, nor the dregs of that
terrible fever, that were to blame for this, but that cruel, pitiless,
ferocious wolf called want. He was dying of the lack of mere
necessaries, and she, miserable woman, was powerless to procure
them; and for this she laid down her head and wept as if her heart
would burst—her passionate sobbing fairly frightened Laurence.
Madeline’s tears were rarely seen; Madeline was always bright,
cheery, almost gay, at the very worst of times; and now came a
reaction, and she was weeping as he had never seen any one weep
before.
“Don’t, Maddie, don’t,” he whispered, feebly stroking her hair; “you
will be better without me, though you don’t think so now. You are
young—only nineteen—many bright days may be in store for you. I
will leave you contentedly, if your father has come home. The
greatest horror I have ever known will be lifted from my mind. You
don’t know, dearest, what torments have racked me as I lay awake
through the long, dark nights, listening to the clocks striking hour
after hour, and wondering what would become of you? Now
Providence has answered the question; your father will give you and
the child a home. There, Maddie, there, don’t; I can’t bear to see you
cry like this; and I—I may get over it, and—— And now, you see, you
have awakened the baby!” as a shrill, querulous cry from the next
room interrupted what he was about to say.
The maternal instinct thus roused, he hoped that her tears would
cease, as he was powerless to arrest them. And Madeline
completely broken down—Madeline, who was always so brave, who
had come out in a new light under the scorching flames of the
furnace of affliction—was a sight that completely unmanned him.
Madeline hastily dried her eyes, strangled her sobs, took her
shrieking offspring out of his cradle, and gave him his midday bottle
—an operation which appeased his appetite and soothed his
feelings. Then she came back to her husband with the child in her
arms, and said in a husky voice, “If you had change of air, good food,
properly cooked, fruit, wine, and the little delicacies all sick people
require, you would get well—I know you would. Promise me, promise
that you will try to get better! Promise me that you will wish to get
better, Laurence,” she continued tremulously, “for—for our sake.”
“I can promise that, at any rate, Maddie,” he answered with a dim
smile; “but you know the old proverb about wishes.”
“And you know that while there’s life there’s hope,” she answered
quickly. “I have hope; you must have hope too! And now I am going
out, you will have to mind baby,” placing the white bundle beside his
father, who eyed his charge dubiously, as it stared at him stolidly,
thumb in mouth.
Madeline hurriedly put on her hat and jacket, and, taking a key,
unlocked a brass-bound desk, and, after a little search, drew out the
morocco case. “Is this it?” she asked, holding it up. “This is what you
mean?”
A nod assured her that it was.
“You would like to look at it once more,” she said, gently laying it in
his hand. “I don’t know how to take it. You are so like her, too,”
looking down at the little oval miniature of a pretty, spirited girl with
dark eyes and dark hair, and seeing her husband’s gaze fixed
greedily on the portrait. “You were so fond of her, Laurence.”
“Not more than I am of you, Maddie,” he answered, closing the
case with a decisive snap. “And my father’s medals,” he said, as he
held them up, and looked at them wistfully. “Well, they will fetch a
few shillings, and they go in a good cause. Here, take them, my
dear, and go, and don’t be long.”
Needless to add this formula. Was she ever long? But time passed
very slowly when Madeline was absent from those two poor attics
which were called home.
CHAPTER VIII.
NOT MARRIED AFTER ALL.
“He has not awoke since, has he?” asked the anxious mother, as,
fully an hour later, she reappeared with a bundle and a basket.
“No”—with a sigh of relief—“I see he is sound,” laying down her load
as she spoke. “And now to begin at the very beginning, and to tell
you everything, Laurence,” opening the basket and producing a
bottle. “Here is some good port wine; I’ve carried it most carefully, so
as not to shake it. You must have a glass at once—that is to be the
beginning.”
“Oh, Maddie, what extravagance! When you——”
“Hush! please to listen,” exhibiting as she spoke a hunch of
grapes, six fresh eggs, a jar of Bovril, and a packet of biscuits from
her seemingly inexhaustible store, and laying them on the table.
“Then you are not going!” exclaimed her husband, in a tone of
deep disapproval.
“Oh yes, I am,” she answered promptly, now opening the bundle,
and shaking out a dress that she had pawned, and regarding it with
an expression that showed it was an old and favourite friend. “Here
is an A B C guide. I go to-night when I have left you comfortable, and
baby asleep. Mrs. Kane’s niece has promised to look after you to-
morrow, and to-morrow night I return, all being well.”
“Then they gave you a good price for the miniature and the
medals.”
“Price!” she echoed indignantly. “They turned the miniature over
and over, and sneered at it, and said they had no sale for such like;
but they could not say that it wasn’t real gold and pearls, and they
gave me eighteen shillings, and said it was more than it was worth,
and ten shillings on the medals. Medals are a drug in the market.”
“Then how—where did you get money for your journey?” asked
her husband, in a tone of amazement bordering on impatience.
“See here!” holding up both her bare hands. Very pretty hands
they were, but now a little coarse from hard work. “Do you miss
anything, Laurence?” colouring guiltily.
“Yes, your—wedding-ring—and keeper,” after a moment’s pause—
a pause of incredulity.
“You won’t be angry with me, dear, will you?” she said coaxingly,
coming and kneeling beside him. “It makes no real difference, does
it? Please—please don’t be vexed; but I got a sovereign on them,
and they are the first things I shall redeem. You must have
nourishing food, even if I had to steal it; and I would steal for you!”
she added, passionately. “I shall only take a single ticket—third
class. Mrs. Harper will surely lend me a few pounds, and I will be
able to leave ten shillings for you to go on with.”
“How can I be angry with you, Maddie?” he replied. “It is all my
fault—the fault of my rashness, thoughtlessness, selfishness—that
you have had to do this, my poor child! Oh, that snowy night was a
bad one for you! I ought to have left you, and walked back.”
“Such nonsense!” cried his wife, whose spirits were rising. “I won’t
hear you say such things! It’s a long lane that has no turning. I think
—oh, I believe and pray—that I do see the end of ours! And now
there is a nice roast chicken for your dinner. I left it with Mrs. Kane
downstairs. She asked me if I had come in for a fortune? A fortune,
indeed! It was only three and three-pence, but I told her that I
believed that I had. Oh dear, oh dear, I hope my words will come
true!”
Madeline’s packing was represented by changing her dress; her

preparations were confined to brushing, rubbing, and inking her hat,
mending her gloves, which, like the typical landlady, “had seen better

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14 MicroRNA–Target Identification: A Combinatorial

Key words MiRNA detection, Biomarkers, Point-of-care setting

RNA molecules are very important regulators of gene expression in

predominately by the RNase III endonuclease Dicer-like1 (DCL1),

2.1 General Detection of miRNAs is challenging because of low abundance,

regulated during cellular processes or in disease conditions. Due to

2.2 Conventional A range of technologies such as Northern blotting, in situ hybridi-

2.2.2 In Situ In situ hybridization involves the use of labelled complementary

2.2.4 RT-qPCR Quantitative reverse transcriptase polymerase chain reaction

2.2.5 Next-Generation Next-generation sequencing (NGS) refers to sequencing technol-

are that it is high throughput, can detect all miRNAs present

2.3.1 Oligonucleotide- Oligonucleotide-templated reactions (OTRs) [31] involve the

engineered to produce optical or electrochemical readouts

2.3.3 Enzyme-Based A range of isothermal enzyme-based signal amplification methods

2.3.5 Electrochemical Electrochemical detection measures an electrical signal produced

2.3.6 Capillary- Capillary electrophoresis [46, 47] is a method to separate biomo-

2.4 Point-of-Care Detection of miRNA in a POC setting to aid clinical decisions

2.4.2 Microfluidic Microfluidic devices process or manipulate small volumes of fluids

The detection of miRNAs is necessary for both a greater under-

miRNA detection techniques to the POC setting for disease treat-

The next morning, leaving Madeline at the station to follow by a

In a very short time Madeline West was Madeline Wynne. She

The very morning after Madeline had despatched her letter, a

Madeline’s packing was represented by changing her dress; her

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