Biology Reference
In-Depth Information
processmultiple samples inparallel. Additionally,
the ideal miRNA detection method should not be
limited due to expensive instruments required or
advanced read-out systems not present in a stan-
dard molecular biology laboratory, and
TABLE 3
miRNA Detection Techniques
General method
Examples
Reference
41
e
47
Hybridization-
based techniques
Microarray-based
analysis
finally,
the method should be reasonably economical.
With a better understanding of the inherent limita-
tions of eachmethod and knowledge of the precise
needs
Northern blot analysis
48,49
RNA-primed array-
based Klenow enzyme
(RAKE) assay
46,50
for
the planned experimental
setup,
researchers will be able to identify
method
of choice for miRNA biomarker discovery. Many
methods for detection and quanti
“
their
”
Invader assay
51
ELISA-based microtiter
assay
52
cation of
miRNAs have been developed, which are brie
y
summarized in
Table 3
. In the following section,
we brie
Bead-based
hybridization assays
53
y describe the most commonly used tech-
niques as well as brie
y discuss the advantages
and disadvantages of their use.
Signal-amplifying
ribozymes
54
Fluorescence-based
single molecule
detection
55
Northern Blot Analysis of miRNAs
The
first method used for miRNA detection
was Northern blot analysis, which is usually
done by denaturing the isolated total RNA
including small RNAs and separating it by urea-
polyacrylamide gel electrophoresis, followed by
transfer of the RNA onto nylon- or nitrocellulose
membranes, crosslinking, and detection of the
bound RNA with a labeled antisense probe. One
of the main advantages of this method is that not
only the mature miRNA of approximately 18 to
25 nt can be detected, but also pre- and sometimes
also pri-miRNAs can be visualized, which might
in some cases be of importance, as the presence
of the precursors, but not the mature miRNA can
be indicative of a potential regulation of one of
the miRNA processing steps or indicate misregu-
lation of miRNA processing in a disease state.
Disadvantages of Northern blot analysis are the
time-consuming procedure, the relatively small
dynamic detection range as well as dif
Splinted ligation assay
56
Gold nanoparticle
probe assay
57
Conducting polymer
nanowires
58
In situ hybridization
59
RNase protection assay
for miRNAs
60
Ampli
cation-
based techniques
PCR-based approaches
61
e
63
Padlock- and rolling
circle ampli
64
cation
T4 DNA ligase
circularization-based
method
65
Dumbbell probe-
mediated rolling circle
ampli
66
cation
culties in
signal quanti
cation. Although protocol modi-
Cloning-based
techniques
Cloning of mature
miRNAs
67
fications like crosslinking the RNA to the
transfer membrane with EDC
49
or the use of
SAGE-based miRNA
serial analysis of gene
expression (mirage)
68
ed oligo probes
48
LNA-modi
exist to enhance
the speci
city and sensitivity of miRNANorthern
blot analysis, the overall sensitivity is rather low,
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