Azure Cielo Real-Time PCR Systems User Manual
Page 40
What is a primer dimer?
A primer dimer is the term used when primers within a reaction interact with each other. This may be
instead of or in addition to the specific target. Many times, within the no template control samples run with
SYBR Green, it is evident that any signal recorded was due to very small products made when the primers
combined to make a nonspecific artifact-the melt curve will show a broad peak (not sharp) with a low
melting temperature (usually 78-79ºC). On a gel for conventional PCR, primer dimers show up as a diffuse
(fuzzy) band at low molecular weight (usually less than fifty base pairs).
If primer dimers are generated, these negative influences will adversely impact the efficiency of the PCR.
The concentration of the primers in the reaction could be reduced or the annealing temperature could be
increased to eliminate this unwanted result.
For my qPCR set up, how much template would be recommended?
A general rule for genomic templates is 50 nanograms of DNA. It is possible to inhibit PCR by adding too
much template to the reaction, so using as little DNA as possible to still get an answer is the best option.
If plasmid template is utilized, very low quantities are needed. Linearized plasmid will amplify better than
circular. Usually, 1 picogram of plasmid DNA can be successfully amplified.
When performing gene expression analysis, the assay will usually load a specific volume of cDNA and not
necessarily a quantity, with the differences normalized to a reference or endogenous control gene.
What is a quencher, and how do I know which quencher to select?
A quencher is a special dye molecule that is attached to the 3’ end of the probe. There are a few different
ones available. Probe providers can make recommendations on the best quencher to use with the fluorescent
dye molecule chosen for detection.
While the probe is intact, the quencher molecule is adjacent to the 5’ end of the probe, where the detection
fluorophore is attached. The quencher will absorb any signal that the fluorophore emits when the two are
in this proximity. Once the exonuclease activity of Taq DNA polymerase cleaves the probe in a sequence-
specific manner during qPCR cycling, the quencher and the fluorophore are no longer held close together. The
quencher is no longer able to absorb the fluorescence emitted, and signal is released. As PCR progresses,
more sequence specific fluorescent signal is freed from the quencher action until the signal reaches threshold
levels and the software assigns a C
t
/C
q
. Signal continues to accumulate, but the important level is that of
reaching above the background.
Cielo specific questions
I have used other qPCR instruments in the past and I never had to do a compensation experiment when I
ran different dye combinations. Why is it necessary on the Cielo to do this? What if I don’t do it?
A multiplex assay can be executed, and data analysis performed without a compensation experiment.
There is a real advantage to running a compensation experiment however, which can make the data
produced more accurate. Any time multiple fluorescent dyes are employed within a single run, cross talk
or spectral overlap is possible. These terms just mean that a small amount of fluorescent signal can cross
channels and can contribute to the signal detected in another dye channel. By running a compensation
experiment, coefficients can be determined which will enable the software to basically filter out any signal
within a given channel that is not actually coming from that channel but bleeding in from a different detection
window. Compensation need only be completed one time for a combination of dyes and the coefficients
generated can be applied to all future runs using that same combination.