Biomedical Engineering Reference
In-Depth Information
SPUD probe (5
μ
M
)(5
-3
):
FAM-
TGCACAAGCTATGGAACACCACGT-(
BHQ1
)
•
•
Real-time thermocycler (e.g. Corbett 6000 or Stratagene MX3005p).
Method
1 Prepare a qPCR master mix sufficient for all RNA samples to be tested in duplicate.
Include a minimum of two 'no RNA sample' controls where the only template is the SPUD
amplicon (SPUD-A). For each RNA sample, prepare 20
μ
l of master mix by combining the
following:
•
0.5
μ
l of water
•
12.5
μ
lof2
×
master mix buffer
•
5.0
μ
lof25m
M
MgCl
2
•
1.0
μ
l of 20 000 copies/
μ
lSPUD-A
•
1.0
μ
lof5
μ
M
SPUD P
•
0.5
μ
l of each of 10
μ
M
SPUD F and SPUD R.
2Add5
μ
l of each RNA or cDNA template to be tested to each of two qPCR tubes.
3 Add 20
μ
l of master mix to each RNA sample.
4 Run in the qPCR instrument using a two-step protocol:
95
◦
C, 10min
1 cycle:
Activation
95
◦
C, 30 s
40 cycles:
Denaturation
60
◦
C, 60 s (collect data)
Annealing/extension
5 Determine the
C
t
value for the control reactions containing SPUD-A only (i.e. no sample
RNA).
6 Determine the
C
t
for reactions containing test samples and compare with the no sample
control.
h
Notes
g
When a two step RT-PCR procedure is adopted it may be preferable to identify only the factors
which inhibit the qPCR and include cDNA (at the concentration desired for subsequent qPCR
reactions) as the test sample.
h
The presence of inhibitors is indicated by a higher C
t
being recorded for test samples than
for the control containing SPUD-A alone. The distribution of the C
t
values from the duplicate
samples containing SPUD-A alone imply the assay coefficient of variance (CV) and this defines
the acceptable range of
b
C
t
values for samples which do not contain inhibitors. Samples with a
C
t
shift greater beyond the CV for the control samples are considered to contain inhibitors, and
should be purified, or a fresh RNA sample extracted.
