I have run a qPCR-assay using a Cy5-probe. On one of the plates, some technical replicates have very high RFU-levels, but the Ct-values of the replicates are almost identical. Does anybody know what might cause these high RFU-levels?
First: Determining the Cycle threshold should be done in the Log scale mode. Using this function reduces the difference in end-rfu (visually). The Linear view can be used for determining the baseline settings when needed. However, you might know this already.
To answer your question, I'd need more information about the qPCR assay.
Is it possible a secondaire target is amplified thus resulting in a higher End-RFU?
This phenomenon could theoretically be used to your advantage. This might be an interesting read; https://www.nature.com/articles/s41598-018-37732-y (Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation)
Or it might be a technical issue with the optical unit of the CFX :)
Hi,
is it a singleplex or a multiplex run? If multiplex I would assume that the sampels with higher RFU are havin no coamplification of another sample whereas the others does.
Sven
Dear Magnhild Erdal , due to my practice I frequently meet with that. As I think, it is not important to receive the same RFU level. More important receive the same Cq values within replicates ). I agree with Maarten van der Salm regarding artefacts or CFX. Usually it is random.
Regarding multiplex, yes different reporters have different RFU in one well in the same time, but as I can see on provided picture it is not multiplex. Am I right?
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