Biology Reference
In-Depth Information
10. From the 6-well plate, passage each clone in triplicate into a
white clear-bottom 96-well plate at 10,000 cells/well and
grow overnight at 37 °C.
11. Remove the media and rinse the cells with 1× PBS (50
μ
L).
12. Lyse the cells by adding 1× Passive Lysis Buffer (25
μ
L) and
shake for 15 min at room temperature.
13. Assay the cells for luciferase expression using a Dual Luciferase
Reporter Assay Kit as described previously (
see
Subheading
3.3
)
and record the luminescence on a microplate reader (
see
Note 8
).
Promising clones obtained from the G418 selections, which have
high fi refl y luciferase readings and low relative luciferase signals,
are further validated by transfection with a miR-122 antagomir
antisense agent to verify that the
Renilla
luciferase expression
responds to a loss of miR-122 function.
3.6 Selection
of a Stable Huh7-
psiCHECK-miR122
Reporter Cell Line
1. Passage each clone into a white clear-bottom 96-well plate at
10,000 cells/well and incubate overnight at 37 °C. Transfect
each clone in triplicate with and without the miR-122
antagomir antisense agent (250 pmol) using X-tremeGENE
siRNA transfection reagent (2
L/well) in Opti-Mem media.
2. Incubate the cells at 37 °C for 4 h. Replace the transfection
media with standard DMEM growth media (without G418).
3. After a 48 h incubation, remove the media, wash the cells with
1× PBS, lyse the cells, and assay with a Dual Luciferase Reporter
Assay Kit as described previously (
see
Subheading
3.3
). The
stable cell line should be chosen based on the level of luciferase
expression and the response to miR-122 antagomir antisense
agent transfection. For the miR-122 antagomir transfection, a
minimum signal to background of 3 is required for a robust
assay [
27
].
μ
To validate that the Huh7-psiCHECK-miR122 reporter cell line
can be used in a high-throughput assay for small molecule inhibi-
tors of miR-122, the cells need to be tested with increasing con-
centrations of DMSO since small molecule libraries are commonly
stored in DMSO as a solvent [
28
,
29
]. In the Dual Luciferase
Assay, the DMSO should have minimal effect on luciferase expres-
sion and cell viability at the concentration to be used for small
molecule screening, typically below 1 % for cellular assays [
28
].
3.7 Effect of DMSO
on the Huh7-
psiCHECK-miR122
Reporter Cell Line
1. Passage the selected Huh7-psiCHECK-miR122 cells at
10,000 cells per well into a white clear-bottom 96-well plate
and incubate at 37 °C overnight.
2. Treat the cells with DMSO (0, 0.1, 0.5, 1, and 2 %) in standard
DMEM growth media and incubate the cells at 37 °C for 48 h.
3. Remove the media, wash the cells with 1× PBS, lyse the cells,
and assay with a Dual Luciferase Reporter Assay Kit as described
previously (
see
Subheading
3.3
).
