An intriguing issue concerning FFPE-DNA is to determine the template amount to be added per amplification reaction, which is particularly important for the evaluation of Q-PCR method efficiency.
This option is favored over simple dilution to minimize inhibition when template amounts are presumed to be minimal.
Standard samples of known template amounts were used to quantify PCR products.
Template amounts ranging from 106 to 100 genome copies per reaction were used for limit of detection determination.
The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA.
Amplification of 18S rRNA was monitored as an endogenous control that was used to normalize template amounts.
A 20-bp segment at the 3' end of the 5 s rRNA gene was amplified as an endogenous control to normalize template amounts.
RT-PCR of PpEF was used as a control to monitor cDNA template amounts.
We initially optimized the assay, since small amounts of non-specific amplicons, salt concentration and template amount may contribute to variation in melt profiles.
At lower DNA template amounts, the culprit's allelic mixture peaks begin to fall below the preset threshold (e.g., 100 rfu).
