Inter run calibration qRT-PCR - (Mar/31/2014 )
i have crawled through the formulas of Hellemans 2007 Paper, and have one or two last questions.
First my sample setup:
I measure 2 GOI and 3HK using the gene-maximation-setup (to much samples for one 96 well plate).
Cq were accepted with a cycle difference of not more tha 0,5. If Cq was over 0,5 i repeated this sample for this gene on one of the following plates.
As IRC i used my positive control for each gen seperately (same cDNA in every run).
The workflow for calculation:
- calculating the arrithmetic mean of Cq-GOI and Cq-Housekeeper in one (???) run
- calculate the Delta-Cq = Cq-HK - Cq-GOI
- calulate the relative quantities RQ = E^(Delta-Cq) (--> RQ still for one GOI in one plate/run??)
- calculate the normalizationfactor NF = geometrical mean of the RQ-Hk (--> how do i calculate this?? is this for one run or all runs??)
- normalization RQ-GOI / NF
- IRC (again geometric mean and division)
In Hellemans it says:
- "IRC´s are identical samples that are testet in both runs"; "It is advisable to use multiple IRCs" and "formula 13'-16' can only be used for interrun calibration if the same set of IRCs is used in all runs to be calibrated": Does this mean for one gene or for the hole experiment? eg I use positive controls for gene x only in cases i measured this gene x on this plate or do i have to measure all positive controls of all genes I will measure in this experiment?
If all this meant i have to use the same set of IRCs (=all positive controls for all genes i will measure in this whole experiment), what is the formula if this is not the case? The paper says, that they are working on it, but i didn´t find a follow up paper...
Thanks for all thoughts and guesses!
Ok i am still a little bit confused.
the relative quantities calculation:
- for my GOI it would be E ^ (Cq-ref.gene - Cq-GOI); per GOI, sample and run
- for my IRC it would be E ^ (Cp-ref.gene - Cq-IRC); per IRC, sample and run???
- and for my reference gene?! Can i take the calculation from GeNorm (E^(Cq-min - Cq))???
I have the same questions from Hellemans's paper on IRC. Have you had any clarification since then?