Biology Reference
In-Depth Information
2.5 Primers
for Cloning
Table 2
Primers for cloning pri-miRNA into pMir-Report Luciferase plasmids
Primer name
Sequence
pri-miR- 16-1 FOR
5′-TGATAGCAATGTCAGCAGTG-3′
pri-miR- 16-1 REV
5′-TGGTCAACCTTACTTCAGCA-3′
Mutagenesis
pri-miR- 16-1 ss_f
5′-TACCACCAGGTAAAAATTGGCGTTAAG
ATTCTAAAATTATC TCC-3′
pri-miR- 16-1 ss_r
5′-CGCCAATTTTTACCTGGTGGTAAGGCA
CTGCTGACATTGCT-3′
2.6 In Vivo
Processing Assay
Luciferase buffers should be aliquoted and stored at −20 °C.
Repeated freezing-thawing cycles of these buffers may decrease
assay performance.
1. 5× Passive Lysis Buffer (Promega). Store at −20 °C. 1× solu-
tion in ddH
2
O can be stored at 4 °C for up to 1 month.
2. Firefly buffer pH (8.0): 25 mM glycylglycine, 15 mM K
x
PO
4
(pH 8.0), 4 mM EGTA, 2 mM ATP, 1 mM DTT, 15 mM
MgSO
4
, 0.1 mM CoA, 75 μL luciferin (
see
Notes 1
and
2
).
3. Renilla buffer pH (5.0): 1.1 M NaCl, 2.2 mM Na
2
EDTA,
0.22 M K
x
PO
4
(pH 5.1), 0.44 mg/mL bovine serum albumin
(BSA), 1.3 mM NaN
3
, 1.43 μM coelenterazine.
4. Luminometer, like GloMax 96 (Promega).
3
Methods
1. One day before transfection seed the cells into two 75 cm
2
flasks. Cell density should reach 50-80 % confluence on the
day of transfection.
2. The day of transfection, replace the medium with 9 mL of
complete growth medium.
3. Allow the transfection reagent to warm up to room tempera-
ture. Pre-warm serum-free DMEM medium to 37 °C.
4. For each flask to be transfected prepare a
transfection mix
by
mixing 8 μg pSUPER-shEGFP (
see
Table
1
for sequences or
8 μg pSUPER-shDROSHA (
see
Table
1
)) with 980 μL serum-
free DMEM. Add 20 μL TransIT-LT1 transfection reagent,
mix well, and incubate for 20 min at room temperature (
see
Note 3
).
5. Dropwise add 1 mL of transfection complexes to the cells and
shake gently by rocking the plate back and forth.
3.1 Transfection
of Adherent Cells
3.1.1 Knockdown of
DROSHA Using shRNA
