Biology Reference
In-Depth Information
4. Ligate the annealed insert with T4 ligase (200 units, 10
L
reaction, 1:10 vector/insert ratio) into the digested psi-
CHECK-2 vector at 4 °C overnight.
5. Transform the ligation reaction into NovaBlue competent cells
and plate on an LB agar plate containing ampicillin (50
μ
μ
g/
mL). Incubate at 37 °C overnight (
see
Note 5
).
6. The construction of the psiCHECK-miR122 vector is con-
fi rmed by sequencing (sequencing primer: 5
′
GCTAAGAA
GTTCCCT 3
′
).
3.2 General
Cell Culture
All cell culture experiments using the Huh7 human hepatoma cell
line should be performed in standard Dulbecco's Modifi ed Eagle's
Medium (DMEM) supplemented with 10 % fetal bovine serum
(FBS) and 1 % penicillin/streptomycin and maintained at 37 °C in
a 5 % CO
2
atmosphere. The Huh7-psiCHECK-miR122 cell line
should be cultured in DMEM supplemented with 10 % FBS,
500
g/mL of G418 (
see
Note 6
), and 1 % penicillin/streptomycin
and maintained at 37 °C in a 5 % CO
2
atmosphere. All Huh7 cell
lines can be passaged using TrypLE Express (trypsin replacement).
μ
3.3 Assessment of
the psiCHECK-miR122
Reporter System
To validate the psiCHECK-miR122 reporter as a sensor for miR-
122, the plasmid is transfected into Huh7 cells, which express high
levels of miR-122, thus leading to a decrease in
Renilla
luciferase
expression compared to the psiCHECK-2 control plasmid. In
order to verify that the
Renilla
luciferase expression is restored by
inhibition of mature miR-122, Huh7 cells are co-transfected with
the psiCHECK-miR122 reporter and a miR-122 antagomir anti-
sense agent.
1. Passage Huh7 cells into a white clear-bottom 96-well plate and
grow overnight at 37 °C until they reach 60 % confl uency.
2. Transfect either the psiCHECK-control plasmid (the original
psiCHECK-2 plasmid containing no known miRNA binding
site) or the psiCHECK-miR122 plasmid (0.5
g) with or with-
out the miR-122 antagomir antisense agent (50 pmol) using
X-tremGENE siRNA transfection reagent (2:1 reagent/DNA
ratio) in Opti-Mem media (200
μ
L total volume) according to
the manufacturer's protocol. Perform all transfections in tripli-
cate for statistical analysis.
3. Incubate the cells at 37 °C for 4 h.
4. Remove the Opti-Mem media and replace with standard
DMEM growth media (200
μ
L).
5. Incubate the cells at 37 °C for 48 h.
6. Remove the media and rinse the cells with 1× PBS (50
μ
μ
L).
7. Lyse the cells by adding 1× Passive Lysis Buffer (25
L; sup-
plied in Dual Luciferase Reporter Assay Kit) and shake for
15 min at room temperature.
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