My two reference genes generate amplicons around 100 bp long, whereas the gene that I have to study produces a 418 bp long amplicon. Different lenghts of amplicons could affect the relative quantification?
If your using taqman probes then efficiency should not be a problem. If not then differences in efficiency between the gene of interest and the housekeeping gene may influence your results.
increasing the amplicon size, exhausts your polymerase enzyme as well.
I will work with sybr. The primers have the same annealing temperature and didn't show any secondary structure by software analysis. But I am considering to design only one primer
As they have different kinetics and also slightly different fluorescence (more SYBR in 400 bp ) i would go for a amplicon in similar size..
Hi Chiara,
The length of the amplicon may affect the efficiency of the reaction. You may fix this by optimizing the concentration of buffer and elongation time. If the reaction has high efficiency with that length of the product, its fine. The best practice is to create an assay with similar efficiencies of endogenous reference and the gene of interest (more than 95%).
Since there is a big difference between your reference and target gene amplicon size, it will be much harder to attempt similar enough efficiency for your target gene. So I will recommend you to go for redesigning the primers with smaller product size.
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