6 replies to this topic

[Jonas Michalko Hamann]

Hi Guys.

 

When comparing different genes or different conditions, would you then use the end RFU or the CT as a measurement? And why or why not? And if the primer dimer risk is considered 0 should this then yield same result?

 

Moved as this appears to be homework.

[bob1]

So - what do Ct and RFU tell you about your sample?

[Jonas Michalko Hamann]

The CT tells me at what cycle i reach a certain fluorescence that is above the baseline thresholdt, and thereby the starting amount of cDNA. The RFU is the total fluorescence, so the amount of PCR product in my sample not considering primer dimer.

[bob1]

Correct - so which one do you use for quantification?   Think about the way PCR works and how would the Ct and/or the RFU alter if you increased the cycle number?

 

Hint - your supervisor doesn't appear to know much about qPCR

[Kongen]

So the Ct value should be independent on the increased number of cycles right, whereas the RFU will increase further and thereby not be a direct measure of what is in the sample. So what is a potential ratio of 1:2 ref and target gene could be seen as as 1:5 after x number of cycles using the RFU because of the higher amplification of more abundant gene.

[Kongen]

However if one would establish standard curves prior to measuring of samples, the two values could both be used right?

[bob1]

So the Ct value should be independent on the increased number of cycles right, whereas the RFU will increase further and thereby not be a direct measure of what is in the sample. So what is a potential ratio of 1:2 ref and target gene could be seen as as 1:5 after x number of cycles using the RFU because of the higher amplification of more abundant gene.

Correct - google delta delta Ct.

 

However if one would establish standard curves prior to measuring of samples, the two values could both be used right?

Only if the efficiency of the reactions are equal, if I have it right.  it's about 8 years since I last did qPCR, so I'm a bit behind on how things work now.