Biology Reference
In-Depth Information
5. Biotek Synergy 4 microplate reader (Biotek) or an equivalent
microplate reader capable of reading luminescence (
see
Note 4
).
6. miR-122
antagomir
antisense
agent:
5
′
ACAAACACCAUUGUCACACUCCA 3
-OMe mod-
ifi ed bases and phosphorothioate linkages. Dissolve to 100
′
with 2
′
μ
M
in ultrapure water.
7. 1× Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 10 mM Na
2
HPO
4
, 2 mM KH
2
PO
4
, pH 7.4, sterilize.
1. 12-well cell culture plates.
2. G418 disulfate: Add G418 disulfate (200 mg) to ultrapure
water (1 mL). Filter sterilize.
3. Cell Titer-Glo Luminescent Cell Viability Assay (Promega):
Prepare the Cell Titer-Glo Reagent according to the manufac-
turer's protocol.
2.4 Determination
of the G418
Concentration
for Selection
of Stable Cells
2.5 Transfection of
the Huh7-psiCHECK-
miR122 Reporter
Cell Line
1. pcDNA3 plasmid (Invitrogen).
2. Restriction enzymes:
Bam
HI and
Bgl
II.
3. 15 cm, 6-well, 48-well cell culture plates.
4. Lipofectamine 2000 transfection reagent (Invitrogen).
5. Cloning cylinders (Corning, Tewksbury, MA, USA).
2.6 Effect of DMSO
on the Huh7-
psiCHECK-miR122
Reporter Cell Line
1. Cell culture grade DMSO: Filter sterilize.
3
Methods
3.1 Reporter
Plasmid Construction
1. Sequentially digest the psiCHECK-2 plasmid (1
μ
g) with
Sgf
I
L
reaction) at 37 °C for 2 h and heat inactivate at 75 °C for 20 min.
2. Separate the digested backbone on a 1 % agarose gel in TBE
buffer at 90 V for 30 min. Excise the digested backbone and
purify using a QIAquick Gel Extraction Kit (see manufacturer's
protocol).
3. Hybridize the insert DNA containing the miR-122 binding
site by combining 50
(10 units, 50
μ
L reaction) followed by
Pme
I (10 units; 50
μ
μ
L of both the sense
(5
CGCAGTAGAGCTCTAGTACAAACACCATT
GTCACACTCCAGTTT 3
′
AAACTG
GAGTGTGACAATGGTGTTTGTACTAGAGCTC
TACTGCGAT 3
′
) and the antisense (5
′
M) and incubate at
90 °C for 5 min followed by cooling to 4 °C over 5 min and
fi nally, incubate at 4 °C for 60 min.
′
) oligonucleotides (1
μ
