Biology Reference
In-Depth Information
The TaqMan
®
probes are designed to be complementary to an
internal stretch of the amplicon (here they are complementary for
the chosen mature miRNA sequence). In addition, these probes
contain a fl uorophore and a quencher covalently attached to the 5
′
and 3
end of the oligonucleotide probe, respectively. The close
proximity of the fl uorophore to the quencher molecule prevents
the emission of fl uorescence. During PCR, the Taq-polymerase
extends the primer and reaches the 5
′
′
end of the probe which is
hydrolyzed by the 5
exonuclease activity of this enzyme. As a
result, the fl uorescent reporter dye is no longer in close proximity
to the quenching group and fl uorescence increases proportional to
the amount of generated PCR products.
′
→
3
′
Unlike mRNAs, mature miRNAs are not polyadenylated. In this
method (Fig.
2
), mature miRNAs are elongated by adding a polyA-
tail to their 3
1.2 Principle
of polyT Adaptor
qRT-PCR
end during the reverse transcription step using
Escherichia coli
PolyA polymerase (PAP). The polyA-tailed miR-
NAs are then converted into cDNA using a modifi ed oligo-dT
primer which also contains a universal tag sequence on its 5
′
end
for subsequent PCR amplifi cation. Although this strategy may be
somewhat less specifi c than the above-described TaqMan approach,
it has nevertheless a number of advantages: (1) Synthesized cDNA
can be used for any available compatible miRNA assay and thus
helps to safe RNA if it is limited. (2) For detection of precursor
miRNA, only an additional specifi c reverse primer targeting the
stem-loop sequence is required (Fig.
2b
). (3) This technology
uses the rather cheap SYBR Green technology to detect the ampli-
fi cation products.
′
The isolation and characterization of homogenous cell populations
are of great importance in order to attribute changes in the bio-
logical state of a tissue, i.e., the miRNA expression to a specifi c cell
type. In most cases, such an attribution is not possible if bulk tissue
is used because the source to prepare the analyte is very heteroge-
nous due to the composition of most tissues containing various
different cell types. Several methods have been developed for the
isolation of homogenous cell populations from within a mixed cell
population; most of them are variations of the so-called laser cap-
ture microdissection (LCM). Manual microdissection is a simple
and cheap alternative to LCM also able to isolate tissues or cells of
interest with suffi cient high precision. We and others routinely use
manual microdissection for our miRNA expression analyses. In
contrast to the LCM, manual microdissection offers greater pro-
tection to biomolecules such as DNA, RNA, or proteins [
4
], likely
because no laser energy is required, for what RNA is particularly
sensitive.
1.3 Microdissection
