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I want to calculate the gene expression of an experiment with the Delta delta Ct method.

I have these results from two runs of a real time PCR (one for the GOI(Gene of Interest) and one for the reference gene ($\beta$ actin) )

Samples | GOI CT | reference gene CT |

1         16.52         16.24
2         18.58         18.47
3         14.55         20.56
wt        23.06         24.17

So to calculate the $2^{-\Delta\Delta CT}$ for my GOI I've done this :

1st - Normalize CTs of GOI to CTs of reference gene

$ΔCT = CT_{sample's} -­ CT_{reference's}$

This new table then occurs:

Samples | ΔCTs 

1         0.28        
2         0.11         
3        -6.01         
wt       ­-1.11 

After this i normalize these ΔCTs to a control sample. And here is where the question was born. On which criteria should i choose the control sample ?

What i've done was to choose the sample with the bigest CT from the first table. And that was the sample No. 2.

2nd - Normalizing all samples to the control sample

$\Delta\Delta CT = \Delta CT_{sample} -­ \Delta CT_{control}$

and the final results are

Samples | ΔΔCTs 

1         0.17        
2         0         
3        -­6.12         
wt       ­-3.08 

Is that method correct ? If yes, let me write once more the question.

On which criteria should i choose the control sample for the last normalization ? Should i choose the sample with the highest Ct or the lowest one ? And why ?

I hope now be more clear.

Question edited to be more clear.

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  • 1
    $\begingroup$ What do you mean by calibrator sample? Standard curve?? See these posts- 1 and 2 $\endgroup$ – WYSIWYG Aug 1 '14 at 6:17
  • $\begingroup$ No. I don't mean standard curve. In the video above, they choose a calibrator sample from target gene samples to normalize later on the ΔCT to it. Here is the video link: youtu.be/tgp4bbnj-ng?t=34s $\endgroup$ – F.N Aug 1 '14 at 10:08
  • $\begingroup$ But what do you mean by calibrator sample.. I have never heard this term.. You have a reference gene which is $\beta$-actin in your case. $\endgroup$ – WYSIWYG Aug 1 '14 at 11:22
  • $\begingroup$ In the video they use the term calibrator sample to mean control case. Nobody uses the term calibrator. Follow good references. Check the link to a paper on RTPCR analysis, provided in the posts that I mentioned in previous comments. $\endgroup$ – WYSIWYG Aug 1 '14 at 11:29

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