Biomedical Engineering Reference
In-Depth Information
4.
Mix the master mix by inverting and gently flicking the tube and following with a
brief pulse spin.
5.
Thaw samples and standards on ice and briefly spin them before use.
6.
Pipet 21
µ
L of the mix into each of the 96 wells in use.
7.
L of standard/sample/water blank in the matching well.
8. Seal the plate using the caps or an adhesive cover and spin the plate at 130
g
for 30 s.
Pipet 4
µ
9.
Insert the plate into the Sequence Detection System and start the run. Make sure
the PCR profile is appropriate. Using the Applied Biosystems Sequence Detec-
tion System, the thermal profile is usually as follows (
see
Note 5
):
2 min 50°C
10 min 95°C
40 cycles of: 10 s 95°C
60 s 60°C
Be sure to change the PCR reaction volume to 25
µ
L.
3.4.3. Analysis of the Real-Time PCR Results
Notice that the Sequence Detection System software may differ between
machines and versions.
1.
Adjust the threshold to the middle of the linear part of the curves and analyze the
amplification curves for the samples and standards. Make sure the standard curve
is linear with correlation >-0.990, that the sample threshold cycle (C
T
) is in
the range of the standard curve, and that the standard deviation (SD) between the
replicates is small; otherwise, repeat the experiment (
Fig. 2
).
2.
The Sequence Detection System software should calculate the Mean Quantity
(the average quantity of your triplicates for each sample according to the stan-
dard curve). This value represents the genome equivalent measurable in your
sample and should be used for further analysis.
3.
The DNA concentration in genome equivalents per milliliter plasma is calculated
using the following formula. It is crucial to use this formula when starting with
different volumes of sample.
C = target concentration in plasma (genome equivalents per milliliter).
Q = target quantity (genome equivalents) calculated by the Sequence Detection
System.
V
DNA
= total volume of extraction (50
L).
V
PCR
= volume of DNA used per PCR reaction (4
µ
µ
L).
V
Ext
= volume of plasma extracted (x mL).
