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Inter run calibration qRT-PCR

IRC inter-run calibration qPCR inter-plate calibration IPC

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#1 Dupasch

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Posted 31 March 2014 - 12:28 AM

Good morning,

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!



#2 Dupasch

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Posted 03 April 2014 - 11:51 PM

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))???

any suggestions??



#3 oyster_lab

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Posted 27 July 2014 - 09:32 PM

Hi Dupasch,

I have the same questions from Hellemans's paper on IRC. Have you had any clarification since then?

Thanks







Also tagged with one or more of these keywords: IRC, inter-run calibration, qPCR, inter-plate calibration, IPC

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