Biology Reference
In-Depth Information
2. Preheat the real-time thermocycler to 57 °C.
3. Transfer 5
L of each Dicer reaction into a pre-cooled, clear
96-well plate on ice. Add 4
μ
L test
compound in the appropriate concentrations to all other
reactions.
4. First add 10
μ
L H
2
O to controls and 4
μ
L
Bst DNA polymerase to each well, mixing thoroughly after
each addition by repeated aspiration. Avoid the formation of
bubbles, as these can disturb fl uorescence measurements.
5. Close the well plate, and place it into the preheated thermocy-
cler. Measure in 2-min increments at 57 °C and
μ
L BRCA master-mix solution, and then add 1
μ
λ
ex
= 485 ± 20 nm
em
= 530 ± 20 nm for 2 h.
6. Calculate the initial linear slopes followed by IC
50
values (
see
Note 12
and Fig.
3
).
and
λ
Fig. 3
Data of a BRCA-based miRNA maturation assay. miRNA maturation with-
out test compound shows the greatest slope (0 % inhibition), and miRNA matura-
tion with denatured Dicer shows no fl uorescence increase (100 % inhibition).
The control where the test compound is added after Dicer incubation shows only
a minor decrease in fl uorescence increase. Error bars represent the standard
deviation of three replicates [
5
]
