Biology Reference
In-Depth Information
Fig.
5
Cleavage with Dicer. Dicer-mediated fl uorescence increase was measured
over the course of 4 h. Incubation of 100 nM beacon with 20 U/ml heat-denatured
Dicer (
left
) and with 20 U/ml recombinant Dicer (
right
)
4. For RP-HPLC (reverse-phase HPLC) we used a “Polaris”
column from Varian Inc. (5
m, 200 Å, 250 mm × 4.6 mm)
heated at 50 °C. You should pay attention on working RNase
free as far as possible during and after HPLC purifi cation.
5. Before analysis on a native gel, the pre-miRNA beacon should
be de- and renatured to form its proper secondary structure,
which enables perfect quenching due to the close proximity of
fl uorophore and quencher. The lower this background signal
appears, the larger the dynamic range and the higher the accu-
racy of the assay.
6. For optical analysis and to test if Dicer can actually cleave the
fl uorescent beacon, measure Dicer-mediated fl uorescence
increase over a certain course of time (Fig.
5
). Use UV/visible
spectrophotometer with ex = 491 nm and a path length of
10 mm (depends on the sensitivity of your spectrophotometer).
Start with pre-miRNA renaturation. Conditions for reaction
are as follows: 25 U/ml Dicer, 1× Dicer buffer, and 100 nM
pre-miRNA beacon. Measure the fl uorescence every 30 min
for 4-6 h.
7. Instead of commercial recombinant Dicer it is possible to use
10 % HEK293 cell lysate as a substitute. Especially for Dicer
activity testing, cell lysate from any other cell line or tissue may
be used. In such settings, we did not observe signifi cant effects
upon the addition of RNAse inhibitor.
8. We used the POLAR star Optima (BMG labtech). Any other
plate reader may be used, but you might have to adjust the
gain factor.
μ
