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requiring large amounts of starting RNA. When
working with radioactive probes, special safety
precautions have to be followed, although the
use of DIG-labeled probes can avoid the use of
radioactive materials. Collectively, these facts
argue against Northern blot analysis for high-
throughput miRNA biomarker detection or
routine analysis for diagnostic purposes.
been developed to overcome this issue. In one
approach, miRNAs in the RNA preparation are
tailed at the 3
0
end by adding adenosine-
residues with poly(A) polymerase. After reverse
transcription with an oligo d(T) primer, the
poly(A)-tailed miRNAs can be analyzed by
quantitative PCR with an miRNA-speci
c
forward and an oligo d(T)-speci
c reverse
primer using SYBR green chemistry.
69
An alter-
native approach uses stem-loop primers for
reverse-transcription that are speci
miRNA Microarray Analysis
Like conventional mRNAs, miRNAs can also
be detected and quanti
c for each
miRNA. After cDNA generation, miRNAs are
quanti
ed by microarray anal-
ysis. For this approach, miRNAs are
uores-
cently labeled and hybridized onto slides
containing probes of all known miRNAs at the
time of manufacturing. After washing away
unspeci
ed by speci
c forward and reverse
uorescent probes.
70
Obvious advantages of PCR-based methods
for miRNA biomarker discovery or adoption
for disease diagnosis or prognosis are the high
sensitivity of this method, requiring only minute
amounts of starting material, and the large
dynamic detection range, reaching up to eight
orders of magnitude. By adjusting the melting
temperature, good ampli
primers and
cally bound miRNAs, signal intensities
are quanti
ed and computer-aided analysis indi-
cates differential miRNA expression of several
hundred miRNAs when compared to different
samples or different conditions. As several steps
of this procedure can be automated, it is less
time- and labor-consuming than other methods,
making high-throughput miRNA pro
cities
can be attained, yielding quantitative informa-
tion on miRNA expression. Other advantages
are the ease of use, the reaction speed, and the
possibility to use multiplexing to analyze several
miRNAs in one miRNA pro
cation speci
ling
possible. Although the microarray technology
is well established and can also detect weakly
expressed miRNAs, it is quite expensive and
only information can be obtained for miRNA
probes that are already present on the chip,
making this method unsuitable for detecting
novel miRNAs. Also, some bias might be intro-
duced into the quanti
ling experiment.
Disadvantages are that only mature miRNAs
are detected in one run (although methods exist
to also quantify the pre- or pri-miRNAs in sepa-
rate experiments) and that only already known
miRNAs can be analyzed, which excludes real
time quantitative RT-PCR analysis for discovery
of novel small RNAs. For validation of miRNA
signatures obtained by, for example, microarray
analysis, small- to medium-sized miRNA
biomarker discovery or clinical diagnostics,
real-time quantitative RT-PCR analysis repre-
sents a very useful tool.
cation, as miRNA hybrid-
ization to the spotted probes depends on the
sequence composition of the miRNA, thereby
making careful design of the probes mandatory.
In general, this method can be applied for the
analysis of known miRNA biomarkers, although
the need for specialized equipment does not
allow use in a clinical setting.
Real-Time Quantitative RT-PCR Analysis
of miRNAs
Due to their small size, real-time quantitative
reverse-transcriptase PCR analysis for miRNAs
is not straightforward. Several approaches have
Deep Sequencing Analysis of miRNAs
In recent years, enormous progress in the
development of novel sequencing technologies
resulted in the advent of several next-
generation sequencing (NGS) machines, which
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