Biology Reference
In-Depth Information
The expression levels of a control miRNA (e.g., miR-21) should
also be measured to further verify specifi city for miR-122.
1. Passage Huh7 cells into a 6-well plate and grow overnight at
37 °C until they reach 60 % confl uency.
2. Treat the cells with the small molecules (10
M fi nal concen-
tration) or the DMSO control (1 % fi nal DMSO concentra-
tion) in triplicate to ensure statistical validity.
3. Incubate the cells at 37 °C for 48 h.
4. Remove the media and wash the cells with 1× PBS buffer
(2 × 2 mL).
5. Isolate the miRNA using a mirPremier microRNA Isolation
Kit according to the manufacturer's protocol.
6. Quantify the miRNA using a Nanodrop ND-1000
spectrophotometer.
7. Perform a reverse transcription reaction with each RNA sample
(10 ng) using a TaqMan microRNA Reverse Transcription Kit
in conjunction with either the miR-122 or miR-21 TaqMan
RT primer (16 °C, 30 min; 42 °C, 30 min; 85 °C, 5 min)
according to the manufacturer's protocol.
8. Using the cDNA products, perform qRT PCR with the
TaqMan Universal PCR Master Mix, No AmpErase UNG and
the corresponding TaqMan miRNA assay probe on a Bio-Rad
MyiQ Real-Time PCR Detection System (1.3
μ
L RT-PCR
product; 95 °C, 10 min; followed by 40 cycles of 95 °C, 15 s;
60 °C, 60 s) according to the manufacturer's protocol.
9. The threshold cycles (
C
t
) can be used to determine the percent
miRNA expression in small molecule-treated cells relative to the
DMSO control, where DMSO represents 100 % expression.
μ
4
Notes
1. The DMEM described here is made from DMEM/HIGH
with
L
-glutamine powder (Hyclone). This growth media can
also be purchased as pre-sterilized liquid media to simplify the
media preparation. In this case, the pH does not need to be
adjusted and only the FBS and penicillin/streptomycin need to
be added.
2. Adjust the pH of the cell culture media before sterilization
using HCl (6 M) or NaOH (2 M) made with ultrapure water.
3. TrypLE Express is a trypsin replacement that is gentle on cells,
maintains healthy cell growth, and achieves faster dissociation.
Trypsin EDTA, which is commonly used in cell culture labora-
tories, can also be used for passaging the Huh7 cell lines.
