How do we explain the information from standard curve in Real Time PCR to say something about the primers....

Question

How do we explain the information that we get from standard curve of different dilution of the target and endogenous control gene in Real Time PCR to say something about the quality of primers and the relation between the target gene and the endogenous control

Answer 1

For your real time PCR to be valid your target and endogenous control should amplify at the same efficiency. Many companies provide primers that have be 'validated' so that they guarantee target and endogenous control primers do amplify at the same efficiency.
If you have made your own primers you will need to validate them. You amplify and work out the delta CT values for each. If they are within the same range (can't remember the exact number)then you can use them.
The difference between your samples for the target gene is then the delta delta CT between samples

http://pathmicro.med.sc.edu/pcr/realtime-home.htm

SUMMARY OF EFFICIENCY(DDCt) METHOD

This method assumes:
minimal correction for the standard gene, or – that standard and target have similar efficiencies.

* The 2 delta-delta Ct variant assumes efficiencies are both 100%

This is an approximation method. But we need to validate that the assumptions are reasonably correct - For this we can do dilution curves to check that the DCts do not change

The only extra information needed for the Pfaffl method is the reference gene efficiency. This involves only a little more work than validating the approximation method

Hope this helps

Answer 2

d'uh?? try this link and scroll to the bottom two thirds:

http://pathmicro.med.sc.edu/pcr/realtime-home.htm

Standard curves show primer efficiency between 0-100%. Should be around 98% at least. 100% efficiency would mean you get a doubling of product at each replication cycle

Answer 3

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Answer 4

Deviation.

Answer 5

We don't.

Is that the royal we?