Biology Reference
In-Depth Information
3.8 Determination
of the Z
Any developed assay needs to be further validated for HTS-
readiness by calculating the statistical parameter Z
, which repre-
sents a quantitative and well-established measure of the quality of
the assay [
25
]. The Z
′
′
factor
factor takes into account the precision of
measuring the maximum and minimum control signals in replicate
wells. The assay should be repeated over multiple days to ensure
reproducibility. A Z
′
factor of 0.5 or above represents an excellent
assay and is suffi cient for high-throughput screening [
25
,
30
,
31
].
′
1. Passage the Huh7-psiCHECK-miR122 cells at 10,000 cells
per well in white clear-bottom 96-well plates. After an over-
night incubation, transfect the cells with the miR-122
antagomir antisense agent (0 or 250 pmol, negative and posi-
tive control, respectively) using the X-tremeGENE siRNA
transfection reagent (2
μ
L/well) in Opti-Mem media as previ-
ously described.
2. Incubate the cells at 37 °C for 4 h. Replace the transfection
media with standard DMEM growth media.
3. Incubate the cells at 37 °C for 48 h, remove the media, wash
the cells with 1× PBS, lyse the cells, and assay with a Dual
Luciferase Reporter Assay Kit as previously described (
see
Subheading
3.3
). Repeat the assay on multiple days to ensure
reproducibility.
4. Determine the Z
= 1 − (3 × SD
positive
control
+ 3 × SD
negative control
)/(Avg
positive
control
− Avg
negative control
) [
25
].
′
factor using the equation Z
′
4
Notes
1. The Dulbecco's modifi ed Eagle's medium (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, penicillin/streptomycin, and G418 disulfate (for selective
media) 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.
4. For the Dual Luciferase Reporter Assay, a microplate reader
with dispensers is preferable for delivering the luciferase sub-
strates; however, the procedure can be adjusted for microplate
readers without dispensers (see manufacturer's protocol).
