Biology Reference
In-Depth Information
luciferase reporter assay for miR-21 function [
13
], demonstrating
that
1
does not inhibit miR-21 function and is not a general inhibi-
tor of the miRNA pathway. Compound
1
also induced a reduction
in both mature miR-122 and primary miR-122 levels, as shown by
quantitative RT-PCR [
14
]. Thus, this pilot screen validated the
ability to discover small molecule inhibitors of miR-122 function.
The steps to identify inhibitors of miR-122 by small molecule
screening [(1) primary small molecule screen in the psiCHECK-
miR122 assay; (2) confi rmation of initial hit compounds by dose
response assays; (3) secondary assays using a psiCHECK-control
reporter to exclude nonspecifi c luciferase activators; (4) secondary
assays using a miR-21 luciferase reporter to exclude general miRNA
inhibitors; (5) qRT-PCR experiments measuring intracellular levels
of miR-122 and other miRNAs] are reported here and can be
applied to identify small molecule inhibitors of any miRNA of
interest using any compound collection after an appropriate
reporter assay has been constructed.
2
Materials
1. Dulbecco's Modifi ed Eagle's Medium (DMEM,
see
Note 1
):
Add DMEM/HIGH with
L
-glutamine powder (11.8 g) and
sodium bicarbonate (3.26 g) to ultrapure water (880 mL).
Mix and adjust the pH to 7.4 (
see
Note 2
). Add fetal bovine
serum (FBS, 100 mL) and penicillin/streptomycin (50× solu-
tion, 20 mL) and fi lter sterilize. Store at 4 °C.
2. DMEM supplemented with 500
2.1 General Cell
Culture
g/mL G418: Add DMEM/
HIGH with
L
-glutamine powder (11.8 g), sodium bicarbonate
(3.26 g), and G418 disulfate (250 mg) to ultrapure water
(880 mL). Mix and adjust the pH to 7.4. Add FBS (100 mL)
and penicillin/streptomycin (50× solution, 20 mL) and fi lter
sterilize. Store at 4 °C.
3. TrypLE Express (Invitrogen): a trypsin replacement (
see
Note 3
).
4. PCR Mycoplasma Detection Set (Takara Bio, Inc., Shiga,
Japan).
5. Huh7 cell line.
6. Huh7-psiCHECK-miR122 stable cell line (Huh7 cells stably
transfected with the psiCHECK-miR122 reporter:
see
Chapter
11
for a discussion of this cell line).
μ
2.2 Primary Small
Molecule Screen
1. White clear-bottom 96-well tissue culture-treated microtiter
plates.
2. psiCHECK-miR122 reporter plasmid (
see
Chapter
11
)
.
3. X-tremGENE siRNA transfection reagent (Roche).
