Hey everyone,
I am currently doing my doctoral thesis at the department of pathology. I designed two different primers for my gene of interest having two different amplification sizes (130bp and 200bp). Now after performing RT PCR (on the same samples and cell lines each) my Ct value differs for each primer (130bp got 30 and 200bp got around 25). Has anyone any explanation for it?
Hi Jan, it sounds to me like the primers and/or conditions targeting the 130 bp fragment might be suboptimal because smaller fragments tend to amplify more efficiently. In other words, you'd expect the 130 bp fragment to have a lower Ct compared to the 200 bp fragment, presuming both primer sets are optimized. But you really need more replicates before drawing any conclusions.
If you are using sybr green as your fluorescent reporter the simple answer is that this is simply an inter-chelating dye. Therefore, longer amplicons have more space for sybr to interchelate. If your threshold is set at the same numeric value then an amplicon that is 33% larger will reach its fluorescence threshold roughly 30% earlier. This is why it is relative qRT-PCR. Unless you are using TAQman based probes you cannot compare Ct values for the same gene using different primers.
- Badges
- Science topic
- Similar topics
- Thesis Research