• High accuracy: high-depth sequencing guarantees that every base is sequenced
many times.
The development of high-throughput NGS methods to study miRNA expression
profiles has exponentially increased the amount of data generated in the field of
miRNA, bringing interesting applications in diagnostic and therapeutic approaches.
Characterization of the entire miRNA spectrum by NGS provides an effective
method to analyse limited sources, such as tumour biopsies from sites of difficult
access, poor quality samples or circulating/exosomal miRNA from biological fluids
[74–77].
3.1.4 Isothermal Exponential Amplification Reaction (EXPAR)
and Advancements
EXPAR is an interesting miRNA analysis method based on isothermal amplification
with high efficiency (10
6
–10
9 -fold). Galas and co-workers developed the EXPAR
method using a combination of polymerase strand extension and single-strand
nicking [78].
The basic reaction involves two steps: miRNAs hybridize with target primes and
extend along the template in the presence of polymerase, and the nicking enzyme
distinguishes the recognition site of double strands to cut off and generate a short
DNA trigger. In the amplification phase, DNA polymerase extends the sequence to
include a nicking enzyme recognition site. A nicking enzyme can then cut one of the
double strands of DNA previously synthetized by DNA polymerase, which produces
additional trigger sequences. This step is repeated several times usually using SYBR
Green as the label. To date, different strategies have been proposed to improve
EXPAR performance. Recently, an advanced miRNA assay based on the two-stage
EXPAR and a single-quantum-dot (QD)-based nanosensor was developed. Importantly, EXPAR products were specifically hybridized with capture and reporter
probes to form sandwich hybrids. These sandwich hybrids are collected on the
surface of 605 nm emission QDs (605QDs), binding to reporter oligonucleotide/
Cy5. Expression of the miRNA of interest can be analysed by Cy5 signals at
excitation wavelength of 488 nm [79]. Another study proposed hairpin probes
which are unfolded through catalysed hairpin assembly with the universal triggers
as the primers [80]. Following a different approach, EXPAR was combined with
high-resolution capillary electrophoresis single-strand conformation polymorphism
(CE-SSCP) analysis. This modified EXPAR method involves the generation of
signal barcodes labelled with a fluorescent dye to simultaneously analyse the
amplified products via CE-SSCP. GeneMapper v4.1 (Applied Biosystems) is the
leading software used to analyse the peak of interest obtained by electrophoretic
mobility [81].
550
C. Dell’Aversana et al.
many times.
The development of high-throughput NGS methods to study miRNA expression
profiles has exponentially increased the amount of data generated in the field of
miRNA, bringing interesting applications in diagnostic and therapeutic approaches.
Characterization of the entire miRNA spectrum by NGS provides an effective
method to analyse limited sources, such as tumour biopsies from sites of difficult
access, poor quality samples or circulating/exosomal miRNA from biological fluids
[74–77].
3.1.4 Isothermal Exponential Amplification Reaction (EXPAR)
and Advancements
EXPAR is an interesting miRNA analysis method based on isothermal amplification
with high efficiency (10
6
–10
9 -fold). Galas and co-workers developed the EXPAR
method using a combination of polymerase strand extension and single-strand
nicking [78].
The basic reaction involves two steps: miRNAs hybridize with target primes and
extend along the template in the presence of polymerase, and the nicking enzyme
distinguishes the recognition site of double strands to cut off and generate a short
DNA trigger. In the amplification phase, DNA polymerase extends the sequence to
include a nicking enzyme recognition site. A nicking enzyme can then cut one of the
double strands of DNA previously synthetized by DNA polymerase, which produces
additional trigger sequences. This step is repeated several times usually using SYBR
Green as the label. To date, different strategies have been proposed to improve
EXPAR performance. Recently, an advanced miRNA assay based on the two-stage
EXPAR and a single-quantum-dot (QD)-based nanosensor was developed. Importantly, EXPAR products were specifically hybridized with capture and reporter
probes to form sandwich hybrids. These sandwich hybrids are collected on the
surface of 605 nm emission QDs (605QDs), binding to reporter oligonucleotide/
Cy5. Expression of the miRNA of interest can be analysed by Cy5 signals at
excitation wavelength of 488 nm [79]. Another study proposed hairpin probes
which are unfolded through catalysed hairpin assembly with the universal triggers
as the primers [80]. Following a different approach, EXPAR was combined with
high-resolution capillary electrophoresis single-strand conformation polymorphism
(CE-SSCP) analysis. This modified EXPAR method involves the generation of
signal barcodes labelled with a fluorescent dye to simultaneously analyse the
amplified products via CE-SSCP. GeneMapper v4.1 (Applied Biosystems) is the
leading software used to analyse the peak of interest obtained by electrophoretic
mobility [81].
550
C. Dell’Aversana et al.