Biology Reference
In-Depth Information
4.5 μL Drosha storage/reaction buffer.
2 μL recombinant His6-Drosha
390-1374
(~2 ng/μL).
1 μL Fe(III) heme-bound NC1 (10× stock).
0.5 μL RNaseOUT (40 U/μL).
1 μL 64 mM MgCl
2
.
1 μL pri-miRNA (~10,000 cpm/μL).
For Drosha-only control, the NC1 stock solution should be
replaced by SEC buffer. For NC1-only control, Drosha stor-
age/reaction buffer should be used instead of the recombinant
Drosha protein (
see
Note 9
).
2. Incubate at 37 °C for 45 min. The reactions generally do not
proceed further after 1 h. Add 10 μL 2× RNA loading dye to
stop the reaction.
3. Analyze the reactions using a denaturing 15 % polyacrylamide
Transcription and purification of pri-miRNAs.
4. Adhere the disassembled gel on the hydrophobic surface of a
Gelbond film (
see
Note 10
). Cover the other side of the gel
with a filter paper. Dry the gel using a gel dryer coupled to a
cold trap and a vacuum pump.
5. Expose the gel to a storage phosphor screen overnight.
6. Scan in the image using a phosphorimager. An example image
is shown in Fig.
3
.
7. Quantify the total intensities of substrate and product bands
using an image analysis program. Background intensities are
subtracted. To calculate the fraction of pri-miRNA processed,
the signals from the pre-miRNAs (
see
Note 11
) are first con-
verted to that of its corresponding pri-miRNA by multiplying
the ratio of U residues in pri-miRNA and pre-miRNA, since the
pri-miRNAs were uniformly labeled using [α-
32
P] UTP. For
example, the ratio is 42/16 = 2.625 for the 150-nt pri-miR-30a
fragment as we used previously [
22
]. The signal of pri-miRNA
processed is then divided by the amount of starting substrate.
4
Notes
1. The reducing reagent DTT (or β-mercaptoethanol) is impor-
tant for keeping DGCR8 active. Make sure that your DTT
stock solution is in a fully reduced state. Solutions of reduced
DTT should have minimal absorbance above 250 nm, while
oxidized DTT has an absorbance peak at 283 nm with an
extinction coefficient of 273 M
−1
cm
−1
[
36
]. We store our DTT
stock solution (1 M) in −20 °C in aliquots and avoid repeated
freeze and thaw.
