Biomedical Engineering Reference
In-Depth Information
—Reagents such as sodium dodecyl sulfate, EDTA, glycerol, sodium pyrophosphate,
spermidine, formamide, guanidinium salts and dimethyl sulfoxide can inhibit reverse
transcriptase (and
Taq
DNA polymerase). Dilute the RNA sample (1 : 10), as this may
dilute out the inhibitor. If this fails, remove inhibitor by ethanol precipitation of the
RNA. Include a 70% (v/v) ethanol wash of the RNA pellet. Ensure all ethanol is
removed, since this will also inhibit downstream reactions. Glycogen (0.2-0.4
μ
g/
μ
l)
can be included to aid recovery of small RNA samples.
—Optimize the PCR primer concentrations;
this
is essential
for optimal assay
efficiency [11].
—Confirm that the annealing temperature is appropriate for the qPCR primers. Most
qPCR assays are designed for an annealing temperature of 60
◦
C, but some primers
may perform better at different temperatures.
—Consider whether the annealing and/or extension times may be too short.
—Optimize the MgCl
2
concentration.
—Consider whether the probe is too long. Probes are usually designed to have an anneal-
ing temperature of 7-10
◦
C higher than primers. In A-T-rich regions, this may lead
to very long probes with inefficient quenching and a tendency to form secondary struc-
tures. Probes are best kept to approximately 35 nucleotides and modifications such as
LNA incorporated to reduce length while maintaining
T
m
.
—Consider whether the probe may have secondary structure by submitting the sequence
to a folding analysis algorithm such as mfold.
—Consider redesigning the qPCR assay. When all possibilities have been explored and
the assay still performs poorly on control targets, it may be that the most effective
solution is to redesign the assay to an alternative region of the gene sequence
•
Consider the possibility that the target mRNA is not expressed in the tissue under analysis.
—Always include the relevant controls to facilitate the troubleshooting process and to be
assured that all data are valid. If the positive control sample is positive then the negative
result for a test sample can be accepted with confidence. Similarly, a negative control
ensures that a positive result in a test sample is more reliable. This is particularly
critical when the positive result is of high
C
q
(i.e. low copy number).
6.3.2 No-template, negative control yields an amplification
product
The master mix may be contaminated with DNA template or PCR product from previous
reactions. Since the products from qPCR are rarely analyzed further, the latter is a relatively
lower risk. However, if this is possible, or if PCR is used to generate products for the standard
curve, it may be advantageous to use a dUTP/thermolabile uracil-
N
-glycosylase-utilizing
protocol. In this case, all PCR reactions are carried out in the presence of dUTP (replacing
dTTP). Prior to amplification of reactions containing native DNA or cDNA (containing T
residues) the reactions are incubated in the presence of thermolabile uracil-
N
-glycosylase.
This digests potential contaminants containing U residues.
