Biology Reference
In-Depth Information
Fig.
4
Example graph from the assay in a 384-well format. Fluorescence increase
upon incubation of 0.5 U/ml Dicer with 20 nM pre-miRNA beacon (
fi lled circle
).
In the presence of an inhibitor (graph shows 100
M kanamycin A,
fi lled square
)
increase is diminished. Controls are heat-denatured Dicer with (
open square
) or
without (
open circle
) inhibitor, which do not show any increase over the indicated
amount of time
μ
6. After incubation, place plate on ice for 10 min before adding
4
l of Dicer enzyme in 1× Dicer buffer with fi nal concentra-
tion of 2.5 U/ml. Final pre-miRNA concentration is 20 nM.
7. Keep on ice until placing plate into plate reader and measure
fl uorescence increase as described above.
μ
4
Notes
1. Labeled RNA strands were purchased from IBA (Göttingen,
Germany). Local suppliers may use other chemical linkers than
those shown in Fig.
2
for the fl uorescein/DABCYL modifi ca-
tions, which probably will not compromise the feasibility of
the assay.
2. This fl uorescence assay or rather the labeled pre-miRNA bea-
con may be considered candidate for real-time miRNA matu-
ration assay and Dicer cleavage assay, respectively, in living
cells.
3. Salt concentration considerably infl uences Dicer activity. For
measuring inhibition of miRNA maturation through binding
to pre-miRNA with certain test substances, physiologic salt
concentration is recommended. In contrast to low-salt condi-
tions, physiologic salt concentration may not be the optimum
for Dicer activity, but outcome reveals more realistic and
closer-to-cell values.
