Biomedical Engineering Reference
In-Depth Information
Notes
bb
This protocol does not include the use of the reference dye ROX; the requirement for a reference
dye is primarily instrument dependent. Refer to instrument manufacturer's recommendations
and if required, adjust the volume of water accordingly.
cc
The volume required to adjust the total volume to 20
μ
l.
dd
MgCl
2
concentration may need to be optimized for different chemistries and for different
reactions. Most reactions using hydrolysis probes work well in a concentration between 3.5 and
6mMMgCl
2
, Molecular Beacons in 3.5mM and Scorpions in 2.5mM.
ee
The volume of the primers required will depend upon the outcome of initial optimisation
experiments; for a pilot test use a 200 nM final concentration. 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 added to the reaction mix and varying primer 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 primer. The primer concentration conditions selected
are those resulting in the lowest C
t
in the absence of primer dimers [11]. In addition, different
probe concentrations may result in different signal intensity and assay sensitivity. After primer
optimisation, identical assays are run including different probe concentrations from 50 to
300 nM [11].
ff
A medium to highly expressed gene will be effectively detected when using the equivalent of
0.5
μ
l of the first strand cDNA (preferably 5
μ
l of a 1 : 10 dilution of the first strand cDNA).
gg
Reactions containing hydrolysis probes are usually performed using a two-step PCR profile.
During this reaction the double-stranded template is melted at 95
◦
C and the annealing and
extension steps both occur during a single incubation at 62
◦
C. Although this is suboptimal
for amplification by
Taq
polymerase, it is believed to encourage more efficient cleavage of the
internal probe, resulting in maximum fluorescent signal per cycle. For optimal amplification
and signal detection, use a three-step protocol for experiments including other probe-based
chemistries.
6.2.7 Quantification methods
There is a wide-ranging debate about which methods are the most appropriate for calcu-
lating PCR efficiency in general and, more specifically, how to determine the efficiency of
individual reactions (http://www.gene-quantification.info). The conventional approach is to
construct a calibration curve by amplifying a target gene of interest in samples consisting
of a serial dilution of template. The template may be cDNA, genomic DNA, PCR prod-
uct or an artificial oligo. A plot of the logarithm of the template concentration (or relative
concentration) against the
C
t results in a linear graph of negative gradient. An assay with
efficient doubling at each PCR cycle should yield a graph of slope
−
−
∼
3.3
cycles to produce a 10-fold increase in PCR product). However, when using this method
it is assumed that the efficiency of the reaction when using the samples as template is
identical to when using the standard template. In order to address this uncertainty there has
been a move to measure each individual reaction by analysis of the entire amplification plot
and attempts to fit a curve which allows predictions about efficiency. Corbett Research has
3.323 (it takes
