Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 24)

18 | SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual 18 |
If your melt profile exhibits a broad peak, this could be due to the presence of a
pseudogene, one product with two melt domains or other target sequence with a similar
melting temperature. Due to the nature of SYBR
® Green, the lack of ability to discriminate
a few base pair differences and very close melting temperatures for two or more
amplicons results in this type of melt profile. Consider the following correction action:
Review the bioinformatics steps taken to design the assay to ensure that all design
considerations were taken into account. Please visit the assay design section in this manual for
further details.
If you suspect the standard curve and dilution points are not within the MIQE guidelines
of 90–110% PCR efficiency with an R
2 of 0.99 or greater, consider the following
corrective actions:
1. Ensure that the standard curve covers at least 5–6 logs of dynamic range. When the
standard curve is too small, the variability of the true efficiency greatly increases.
2. If the R 2 is <0.98, review the standard curve data points for outliers. Remove any outliers
where the ∆Cq is >0.5 for the group. For example, if your 100 pg dilution point has Cq
values of 29.2, 29.6, and 30.5, you should remove the Cq value of 30.5. If there are too
many outliers, it may be a sign of other technical issues.
3. Evaluate the most dilute points of the standard curve to ensure they are not out of range.
In Figure 17, you can see that the most dilute points are yielding Cq values similar to
less dilute points. This is usually due to primer dimers, which can be resolved through
primer concentration optimization (see the primer matrix discussion on page 17 for
more information).
Fig. 16. Broad melt curve profile where potentially more
than one product or pseudogene exists around the same
melting temperature.
Melt Peak
Temperature, °C
–d
(R
FU
)/d
t
90 80 70 60
500
400
300
200
100
0