Biology Reference
In-Depth Information
3.3 Confi rmation of
Initial Hit Compounds
by Dose Response
Compounds which elicit RLUs that are greater than the threshold
X
+ 3 ×
= standard deviation) need to be re-assayed in
triplicate to confi rm the validity of the hit compound. In addition,
Huh7-psiCHECK-miR122 stable cells should be treated with
potential hit compounds in increasing concentration to test if a
defi ned dose-dependent response can be established.
σ
(
X
= mean,
σ
1. Passage Huh7-psiCHECK-miR122 cells into a white clear-
bottom 96-well plate at 10,000 cells/well and grow overnight
at 37 °C.
2. Replace the growth media with standard DMEM media
(100
L) supplemented with the small molecules (0.1, 0.5, 1,
5, and 10
μ
M fi nal concentration) or a DMSO control (1 %
DMSO fi nal concentration) in triplicate.
3. Incubate the cells at 37 °C for 48 h.
4. Remove the media, wash the cells with 1× PBS, lyse the cells,
and assay with a Dual Luciferase Reporter Assay Kit as previ-
ously described (
see
Subheading
3.2
).
5. 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.
6. From the RLU data, generate a dose-response curve and cal-
culate EC
50
values, the concentration that gives 50 % of the
compound's maximal response [
22
,
23
].
μ
3.4 Secondary
psiCHECK- Control
Assay
When using a luciferase-based reporter assay, it is possible for
small molecules to increase the luciferase signal in a nonspecifi c
fashion leading to false-positive hits [
24
,
25
]. Therefore, putative
hits which induce a signifi cant increase in the relative luciferase
signal should be assayed in Huh7 cells transfected with the
psiCHECK-control plasmid to validate their activity as miR-122
inhibitors and confi rm that they do not increase the luciferase sig-
nal in a non-miRNA-specifi c fashion. In the presence of the psi-
CHECK-control vector, there should be no statistically signifi cant
change in the normalized
Renilla
luciferase signal for the small
molecule-treated cells.
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-control 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.
