Biology Reference
In-Depth Information
RNA for the generation of an miRNA library. A
drawback of NGS analysis is long instrument
run times of up to one week and the cost of
kits for library generation and running the
NGS machines, which have on the other hand
declined sharply in the last years. Analysis of
the large data sets of several gigabytes requires
the necessary hardware and some bioinformatic
computer skills, though software to aid in the
analysis is increasingly being developed. NGS
technology enables researchers in large-scale
approaches to identify known and potentially
novel miRNAs as well as other small RNA
classes, which could also be used as biomarkers
for diagnostic or prognostic use.
temperature and salt or detergent concentration,
nonspeci
c binding can be reduced. Utilization
of LNA-modi
ed probes can also lead to
increased speci
city and sensitivity. Drawbacks
are the slow analysis speed and dif
cult quanti-
fication. In addition, the a priori detection of new
miRNA biomarkers is not possible with this
technique.
Other Methods of miRNA Detection and
Quantification
Since the year 2000, many different additional
methods for miRNA detection and quanti
ca-
tion have been developed, some of which are
summarized in
Table 3
. Excellent overviews of
these methods are given by Wang and Yang
73
and Hunt and colleagues,
74
among others. In
summary, a large variety of methods can be
used for miRNA detection and quanti
In Situ
Hybridization of miRNAs
Like for other RNA molecules, in situ hybrid-
ization can also be applied for miRNA detection
and localization. With this method, labeled RNA
probes allow localization of a speci
cation.
When performing experiments for the discovery
of differentially regulated miRNAs or the identi-
c miRNA in
a tissue section or also in entire animals (whole
mount in situ hybridization) as in developing
zebra
fication of miRNAs that can serve as novel
biomarkers, advantages and disadvantages of
each considered method need to be weighed.
sh embryos. Probes can either be labeled
with radio-,
fluorescent-, or antigen-labeled
bases and are then localized and quanti
ed in
EXAMPLES FOR MIRNA
BIO
MARKER DISCOVERY STU
DIES
situ using either autoradiography,
uorescence
microscopy, or immunohistochemistry. Using
two differently
fluorescently labeled probes,
two independent miRNAs can be detected
simultaneously by
nition of a biomarker being
a characteristic that is objectively measured and
evaluated as an indicator of normal biologic
processes, pathogenic processes, or pharmaco-
logic responses to a therapeutic intervention still
holds true. When thinking of biomarkers in
academic research settings, cancer medicine in
particular comes into mind. Here, biomarkers
are seen as being either diagnostic of tumor
biology or prognostic of disease or therapeutic
outcome. A biomarker can be even more than
this: it could be helpful when trying to under-
stand unclear disease symptoms, and it can
improve diagnosis and treatment by detecting
early-stage disease, choose the best therapy, or
identify patients with high risk of relapse. In
The classical de
fluorescence in situ hybridiza-
tion (FISH).
Fixation of sections with, for example, para-
formaldehyde
fixes the target miRNAs in place
and increases the access of the probe. Cryosec-
tions are generated from frozen tissue blocks
and stored on microscope slides, which are
subsequently hybridized with the labeled
miRNA probes at elevated temperatures. After
washing away excess probe, probes potentially
need to be developed and the tissue sections
can be analyzed. In situ hybridization is a very
sensitive method and allows the cellular and
subcellular detection of miRNAs. By adjusting
solution parameters
such as hybridization
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