What cycle is best for analysing data? - (Aug/21/2015 )

For example if my qpcr protocol is 50 cycles, should I be using the RFU that is at 50 cycles or a lower cycle number e.g. cycle 40? Would you likely end up with a different outcome if you were to compare the RFU of the last cycle versus one that was about 10 cycles before?

Thanks

Vyse on Fri Aug 21 17:31:40 2015 said:

For example if my qpcr protocol is 50 cycles, should I be using the RFU that is at 50 cycles or a lower cycle number e.g. cycle 40? Would you likely end up with a different outcome if you were to compare the RFU of the last cycle versus one that was about 10 cycles before?

 

Thanks

Hi,

I don't understand what is RFU. Secondly, you should have qPCR for 40 cycles at max. If there are products >35 cycles then you shouldn't consider them. Any Cq values between 20-30 are considered good for statistics. Hope that answers your question.

Mad Researcher on Fri Aug 21 18:48:22 2015 said:

 

Vyse on Fri Aug 21 17:31:40 2015 said:

For example if my qpcr protocol is 50 cycles, should I be using the RFU that is at 50 cycles or a lower cycle number e.g. cycle 40? Would you likely end up with a different outcome if you were to compare the RFU of the last cycle versus one that was about 10 cycles before?

 

Thanks

Hi,

 

I don't understand what is RFU. Secondly, you should have qPCR for 40 cycles at max. If there are products >35 cycles then you shouldn't consider them. Any Cq values between 20-30 are considered good for statistics. Hope that answers your question.

 

RFU is basically an arbitrary figure for the respective level of fluorescence generated in a qPCR reaction (the stuff on the Y axis).

In fact the analyse Ive used in the past I have used a combination of the RFU at cycle 50 with the CP valve to guage + from - reactions. I assume if I was just coming to conclusions using just the RFU at cycle 50 that this would be a bad idea.

So I guess what I was trying to ask was would I see much difference in end result between analysing using: RFU at cycle 50 + CP value cutoff between 20-30 v RFU at cycle 40 + CP value cutoff between 20-30?