Biomedical Engineering Reference
In-Depth Information
5 Obtain a melting profile (as below, or according to the thermocycler manufacturer's
instructions)
aa
95
◦
C, 1min
1cycle:
55
◦
C, 30 s
40 cycles:
Collect data
Repeat and increase the temperature by 1
◦
C per cycle
Collect data
Notes
u
Most commercially available blends also contain MgCl
2
, but some reactions benefit from
optimizing the MgCl
2
concentration.
v
This protocol does not describe the use of a reference dye such as ROX, but such a reference dye
may be included in the master mix. Many real-time thermocyclers require a constant reference
dye in order to normalise optical differences in the detection of fluorescence from each well.
The dye is included in the master mix and is detected by the instrument. If this is required
(refer to instrument manufacturer) and is not contained in the 2
×
master mix buffer, adjust
the volume of water accordingly.
w
Adjust the volume of water according to the volume of primers and template used. In this
example, the primers are at a final concentration of 200 nM each, and 5
μ
l of the template is
used.
x
The volume of the primers required will depend upon the outcome of initial optimisation
experiments; for a pilot test use a final concentration of 200 nM. Most assays are more
efficient and sensitive when run under optimal conditions, and primer concentration greatly
influences assay performance. The protocol described above can also be used to optimise
primer concentration. In this case, cDNA is addedtothereactionmixandvaryingprimer
concentrations are added to each reaction. It is advisable to select primer concentrations
between 100 and 300 nM and test all combinations of forward and reverse primers. The primer
concentration conditions selected are those resulting in the lowest C
t
in the absence of primer
dimers [11].
y
When first strand cDNA is used, a target that is believed to be expressed at a medium to high
level will be detected using 5
μ
l of a 1 : 10 dilution of the RT reaction.
z
In most cases the annealing temperature for the primers is designed to be 60
◦
C. If this is not
the case adjust the annealing temperature component of step 2.
aa
The melt curve protocol is usually set automatically using the appropriate selection from the
thermocycler software. Alternatively, a series of incubations are created such that the reaction
is held for 30 seconds at increasing temperatures. The melt would begin by holding the PCR
product at the start temperature, for example 55
◦
C. After 30 seconds the temperature would
be increased to 56
◦
C and the products held for 30 seconds. This would be repeated until an
incubation temperature of 95
◦
C is reached.
6.2.6 qPCR using labeled oligonucleotide probe detection
As an alternative to using DNA binding dyes, probe-based assays using reporter oligonu-
cleotides may be used for amplicon detection (see Protocol 6.7). These probes are labeled
oligonucleotides that are targeted towards the amplified target. At very low (
<
1000 copies)
target concentration there is a greater probability of non-specific amplification and problems
