Hello,
I‘m working with ready made primers for 3 genes (one of them is GAPDH- the housekeeping gene) in taqman gene expession assay. The GAPDH primer has VIC dye while the other two primers have FAM dye, but the results of real time PCR are somewhat weird! The two primers give readings (graphs) for both dyes while they only have FAM. Is this normal? Or do I need to adjust something?
I attached a photo for the graphs I got, where A & B are the genes I’m analyzing and G is the GAPDH.
Would be grateful for any help.
Thanks in advance.
Given the obtained CTs on the right graph (A: 32; B: 30; G: 25), I think that your multiplex is working. You can have a better visualization on linear mode (instead of algorithmic mode) and down the baseline a bit.
Thank you Dr. Aissa Saidi for your response,
I’m not doing a multiplex assay. I’m analyzing each gene separately (in a separate tube).
That’s why I’m confused. I don’t know if it is normal. But to my understanding I guess each assay should give only one graph.
Would be glad to hear your opinion.
I don't know what you have in the recation mix, but if you have only one PCR set (1 probe + 2 correspondant primers) in a tube you should have only one curve. Nevertheless, GAPDH (HKG/IPC) is used to validate the reaction in a duplex (or multiplex) PCR.
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