Biomedical Engineering Reference
In-Depth Information
12.
Reaction volumes between 20 and 50
µ
L had been tested. The system will work
with 20
L works well, but
it is not always necessary to use such a large volume. Very small differences in
collected data might occur if the reaction volume is different from the volume
that the assay is characterized for.
µ
L, but the deviations are increased. A volume of 50
µ
13.
If the concentration of the DNA is too low, up to 4
L Chelex-extracted solution
can be used. If the concentration is still too low, the DNA solution must be con-
centrated by, for example, evaporation, precipitation, or extraction into a smaller
volume using a kit.
To save DNA and for highly concentrated samples, smaller volumes or dilutions
in water can be used.
µ
14.
Higher primer concentrations result in a decrease of the reaction efficiency in the
multiplex assay. This can be attributed to an increase of unspecific reactions. In
addition, excessively high concentrations of genomic DNA are detrimental to the
reaction.
M
also work fine, but a little accuracy may be lost,
especially at the threshold of 0.675.
16. Sample setup on ice is important in order to minimize nonspecific product forma-
tion. The reaction plate should be kept on ice at all times prior to starting the real-
time PCR.
15.
Probe concentrations of 100
µ
17.
Run the program always at the same heating and cooling rates. Differences in
the temperature program will result in slight differences of the final C
T
-
and
∆
C
T
-values. We use the “emulation off” setting because it is faster.
18.
The baseline setting is very important in order to achieve optimal results in the
analysis. It is used to separate unspecific fluorescent signal from the signal gen-
erated by the reaction. If the baseline is too far away from the amplification curve,
a significant proportion of the results, especially those with lower thresholds,
will be adversely affected by noise.
Setting the baseline too close will skew the amplification curves and change the
∆
C
T
to smaller values.
19.
In the amplification plot view, check whether the amplification curves of both
dyes look normal and parallel for all three replicates. If the three replicates do not
look very similar, something may be wrong with at least one of the reactions. This
can be owing to contamination with DNA (pipetting errors), dirt or fluorescence
source other than the dyes, leakage of the well, presence of an inhibitor or other
reasons. If the curves do not resemble each other closely, the test should be re-run.
20.
After 40 cycles of amplification, the
∆
R
n
will usually be between 2.0 and 3.0.
R
n
of less than 1.5 are generally of poor quality.
If using dual-labeled probes, the
Runs with a final
∆
R
n
will be smaller than when using MGB
probes. This is a result of less efficient quenching and resulting higher back-
ground fluorescence in the longer dual-labeled probes.
∆
21.
The use of several thresholds increases the accuracy of the method. By using the
four proposed thresholds, fluorescence data can be collected during three or four
cycles; compare with the use of one threshold, in which case data is collected
from only two cycles. A threshold of 0.1 sometimes contains a good amount of
