As long as I know FAM is one of the brightest used fluorophore molecule when making a qPCR test. So why Am I getting this low RFU fluorescence mixed with kind of the normal signal I would get from using this reporter dye? is it because the probes were damaged or degradated? or is it an inhibition case scenario Im leadding with? internal controls did not amplify correctly
I would like to ask as well why is background noise fluorescence normaly produced during a qPCR reaction? is it because fragmentation of some probes that fluorescence is emitted or just the signal caught by amplification of some initial genetic product?
I'll be really glad with your answers, thank you very much!!
Yes, in general FAM (shorter wave length in real time dyes) provides much better intensity than any other probes
There are so many reasons to get these kinds of amplification pattern in real time PCR. If this probe set is routinely used and if you get very good RFU, then only we can think about degradation of probes. Otherwise, we need to start from several basic points like PCR optimization, primer-probe concentration, annealing temperature, wavelength chosen for reading, height of the channel (if manual mode is applied instead of in-built dynamic modes) duration of data collection (in seconds) etc. I hope, you can start from the point whatever applicable to you.
I also got similar low RFU in a multiplex PCR in FAM/HEX/CY5 probe combination. After proper optimization, we never faced such scenario...
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