Biology Reference
In-Depth Information
1.1 Principle of
Stem-Loop qRT-PCR
The stem-loop oligonucleotide design introduced by Applied
Biosystems is able to specifi cally reverse transcribe only the selected
mature miRNA which is subsequently amplifi ed by qRT-PCR
(Fig.
1
) [
3
]. A typical stem-loop reverse transcription (RT) primer
includes a 5-8 nt 3
end of the mature miRNA, a stem and a loop sequence which also
contains a universal 3
′
overhang sequence complementary to the 3
′
priming site for the subsequent qRT-PCR
step. The idea behind the stem loop design is that base stacking
and spatial constraint of the stem-loop structure improves specifi city
and sensitivity of the assay. Furthermore, it may also prevent bind-
ing of the RT primer to double-strand genomic DNA molecules,
thus RNA sample preparation can be omitted. Lastly, stem-loop
RT primers can be used for multiplex RT reactions. Apart from the
requirements of a miRNA-specifi c stem-loop oligonucleotide for
RT, the subsequent PCR steps include only standard qRT-PCR
technology. A specifi c feature of the herein described TaqMan
®
miRNA qRT-PCR technology from Applied Biosystems is the use
of the so-called TaqMan
®
probe which lends higher specifi city
to the assay than conventional qRT-PCRs without this probe.
′
Fig. 1
Schematic description of TaqMan-based real-time quantifi cation of miR-
NAs. This technique includes two steps: stem-loop RT and real-time PCR. Stem-
loop RT primers bind to the 3
portion of miRNA molecules and are reverse
transcribed. Subsequently, the RT product is quantifi ed using conventional
TaqMan PCR that includes miRNA-specifi c forward primer, reverse primer, and a
dye-labeled TaqMan probe (fi gure adapted from ref.
3
)
′
