Biology Reference
In-Depth Information
5. Record the Renilla luciferase activity measurement by adding
Renilla buffer to the well.
1. Luciferase assay results are normalized as follows:
3.4 Evaluation
of Results
Fireflyluciferase activity
Renilla lucifer
Normalized activity
=
aseactivity
2. The processing efficiency of pri-miRNA construct can be
calculated:
normalized activity of wt cons
truct
normalized activity of ss construct
pri miRNA processing efficiency
̺
=−
1
In this calculation the processing efficiency of the ss construct
will be equal to 0.
4
Notes
1. Dithiothreitol (DTT) is highly unstable in water solutions,
aliquot and store at −20 °C, and thaw the aliquots on ice.
2. The addition of Coenzyme A (CoA) allows a more sustained
plateau of activity facilitating subsequent analysis. CoA in
micromolar concentrations produces a more intense and
sustained light emission because of the more favorable kinetics
of the reaction of the enzyme with the luciferyl CoA substrate.
In the presence of CoA, the luciferase assay yields stabilized
luminescence signals with significantly greater intensities.
3. Transfection reagent should be added at the end of the master
mix preparation.
4. The reaction times can vary for different cell lines. The proto-
col should be optimized before starting the experiments.
5. Depending on the reagent used for transfection it might be
necessary to change the medium at different time points.
Follow the manufacturer's instructions when transecting cells
with a different reagent.
6. The amount of plasmids and reagent used for the experiments
needs to be optimized for every cell type.
7. Presence of detergents in the lysis buffer (i.e., TritonX)
contributes to increase in the intensity and duration of light
emission. However, some types of nonionic detergents com-
monly used to prepare cell lysates (i.e., Triton X-100) can
intensify coelenterazine autoluminescence.
8. The stability of FF luc has been shown to be 6 weeks at 4 °C in
cell lysates.
