Biology Reference
In-Depth Information
4. Replace the Opti-MEM media with standard DMEM growth
media (100
M)
or a DMSO control (1 % DMSO fi nal concentration) in
triplicate.
μ
L) supplemented with the small molecules (10
μ
5. Incubate the cells at 37 °C for 48 h.
6. 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
3.2
).
7. Calculate the ratio of
Renilla
to fi refl y luciferase expression for
each well to provide relative luciferase units (RLUs). The aver-
age and standard deviations for each of the triplicates is calcu-
lated from the RLU values.
3.5 Secondary
miR-21 Luciferase
Assay
Compounds that increase luminescence in a miRNA-specifi c fash-
ion could be specifi c inhibitors for miR-122 or could inhibit a gen-
eral component of the miRNA pathway. In order to exclude general
miRNA pathway inhibitors, the small molecules should be assayed
with a luciferase reporter assay for a different miRNA. Here, a
miRNA-21 reporter assay in Huh7 cells is used to validate the
specifi city of the miR-122 inhibitors, although other miRNAs
could be used as control targets as well.
1. Passage Huh7 cells into a white clear-bottom 96-well plate and
grow overnight at 37 °C until they reach 80 % confl uency.
2. Transfect the Huh7 cells with the psiCHECK-miR21 plasmid
(0.5
g) using the X-tremGENE siRNA transfection reagent
(3:2 reagent/DNA ratio) in Opti-Mem media (200
μ
μ
L total
volume) according to the manufacturer's protocol.
3. Incubate the cells at 37 °C for 4 h.
4. Replace the Opti-MEM media with standard DMEM growth
media (100
μ
L) supplemented with the small molecules
M fi nal concentration) or a DMSO control (1 % DMSO
fi nal concentration) in triplicate.
5. Incubate the cells at 37 °C for 48 h.
6. 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
3.2
).
7. Calculate the ratio of
Renilla
to fi refl y luciferase expression for
each well to provide relative luciferase units (RLUs). The aver-
age and standard deviations for each of the triplicates is calcu-
lated from the RLU values.
(10
μ
The activity of the small molecule inhibitors can be further ana-
lyzed by quantitative Real-Time PCR (qRT PCR) to measure
their direct effect on miR-122 expression levels in Huh7 cells.
3.6 Quantitative
RT PCR
