Biology Reference
In-Depth Information
4. miR-122 antagomir antisense agent: 5
′
ACAAACACCAUU
GUCACACUCCA 3
-OMe modifi ed bases and phos-
phorothioate linkages. Dissolve to 100
′
with 2
′
M in ultrapure water.
5. Opti-MEM Reduced Serum Medium (Invitrogen).
6. Compound library in a 96-well format at a working stock dilu-
tion of 1 mM in 100 % DMSO. For example, the Diversity Set
II small molecule library from the NCI Developmental
Therapeutics Program.
7. Cell culture grade DMSO: Filter sterilize.
8. Dual Luciferase Reporter Assay Kit (Promega): Prepare 1×
Passive Lysis Buffer, Luciferase Assay Reagent II, and Stop &
Glo Reagent according to the manufacturer's protocol.
9. Biotek Synergy 4 microplate reader (Biotek) or an equivalent
microplate reader capable of reading luminescence (
see
Note 4
).
10. 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, sterilized.
μ
1. psiCHECK-control plasmid: the psiCHECK-2 plasmid con-
taining no known miRNA binding site (Promega, Madison,
WI, USA).
2.3 Secondary
psiCHECK- Control
Assay
1. psiCHECK-miR21 reporter plasmid (the psiCHECK-2 plas-
mid containing a miR-21 binding site in the 3
2.4 Secondary
miR-21 Luciferase
Assay
′
UTR of the
Renilla
luciferase gene).
1. 6-well tissue culture-treated plates.
2. mirPremier microRNA Isolation Kit (Sigma Aldrich, St. Louis,
MO, USA).
3. Nanodrop ND-1000 spectrophotometer.
4. TaqMan microRNA Reverse Transcription Kit (Applied
Biosystems, Carlsbad, CA, USA).
5. TaqMan miRNA assays for miR-122 and miR-21(Applied
Biosystems).
6. TaqMan Universal PCR Master Mix, No AmpErase UNG
(Applied Biosystems).
7. Bio-Rad MyiQ Real-Time PCR Detection System (Bio-Rad,
Hercules, CA, USA).
2.5 Quantitative
RT-PCR
3
Methods
All cell culture experiments using Huh7 cells should be per-
formed in standard DMEM supplemented with 10 % FBS and
1 % penicillin/streptomycin and maintained at 37 °C in a 5 %
3.1 General Cell
Culture
