Biology Reference
In-Depth Information
Fig.
3
Analysis of ligation product (pre-miRNA beacon) with 20 % gels.
(
a
) Denaturing PAGE of ligation reaction with the mixed fragments (
1
) and the
ligation product (
2
). (
b
) Native PAGE of ligation without staining. FAM-36-mer (
1
),
DABCYL-36-mer (
2
), mixed fragments (
3
), ligation product (
4
). (
c
) Same as in
(
b
) but stained with fl uorescent dye which detects ligation product (
4
) that is
quenched
3.3 Preparation
of Pre-miRNA Prior
to Fluorescence
Assaying
1. Prior to use in the assay de- and renature labeled pre-miRNA
at a stock concentration of 200 nM in 1× Dicer buffer by heat-
ing for 2 min at 95 °C and cooling to room temperature (rt)
for over 30 min.
2. Test if Dicer can actually cleave prepared pre-miRNA beacon
(
see
Note 6
).
1. For Dicer cleavage assay prepare reaction mixture in one well
in a 384-well microtiter plate on ice. Reaction mixture with
total volume of 40
3.4 Fluorescence
Assay
l per well contains 20 nM labeled pre-
miRNA, 1× Dicer reaction buffer, and 2.5 U/ml Dicer enzyme
or heat-denatured Dicer for negative control (
see
Note 7
).
2. Place the plate into plate reader, and measure fl uorescence
increase every 1-10 min for 4 h with ex = 491 nm, em = 520 nm,
and gain = 2,500 (
see
Note 8
).
3. Analyze fl uorescence increase from the slope of the linear
region that occurs in the range of 5-50 min (Fig.
4
). For avoid-
ing false-positive results, do a control using RNase inhibitor
(
see
Note 9
).
4. For testing of potential pre-miRNA binders (test substances)
combine 32
μ
μ
l of a 25 nM pre-miRNA solution in 1× Dicer
buffer with 4
μ
l test substance in one well in a 384-well microti-
ter plate.
5. Cover with parafi lm, and incubate for 30 min at rt.
