What to choose as a calibrator? - When looking at basal gene expression (May/04/2006 )

Hi All-

Does anyone have any suggestions on what to use as a calibrator for relative RT PCR if I just want to look at basal gene expression, and not fold induction?

I just saw a presentation where they did not use a calibrator, but instead just looked at Ct difference between the HKG and the GOI. However, my supervisor thinks this is a poor way to present data. We are thinking now about using gene expression in fresh liver tissue as the calibrator, and then presenting the data by showing fold change in expression after plating primary hepatocytes. But is there a better way to do this? I am concerned that gene expression after plating will decrease so much compared to the fresh tissue that they will be in different dynamic ranges for PCR...?

Or do you think I should just use a standard curve for this experiment?

Thanks for your time and suggestions!

we use actin and gadph

People need to realise the difference between a reference/house keeping gene (HKG) and a calibrator sample or tissue used in the delta delta Ct method of analysis. I think soluene is talking about the latter.

I've been using the pfaffl method, 2001 which uses a single sample within your run (usually the highest expressing) as a calibrator to which everything else is expressed relatively. Would this fit into your set up?

The calibrator can really be anything since you are using a relative method it's not the actual numbers that matter just their ratio to each other.

Hope this helps

Thanks neuromatt - you are right, I was not referring to the HKG.

That is a good idea, using the highest expressing gene as the calibrator. I will give that some more thought.. Thanks!

I'm a bit confused, please help me understand!
The reference gene is used to normalize each sample, right?
I thought that the reference gene is used in Pfaffl's method...?
Do you use a calibrator instead of reference, or both?

Hi Britt-

The reference gene is used to determine the dCt. You subtract the Ct of the reference gene from the target gene for all samples.

The calibrator is what you use to determine the ddCt. You subtract the dCt of the treated or test samples from the dCt of the calibrator (typically, untreated controls or Time 0 time point).

My problem above is that I didn't have any treated vs. untreated - only untreated - in which case I was not sure what to use as a calibrator to get the ddCt.

I hope that helps!

Yes, now it's perfectly clear! I'm always looking on treated vs untreated, not just using the word 'calibrator'. Thank You!