3 replies to this topic

[George250]

Hi to all,

I am new in QPCR and I have some questions.. Last week I did my first QPCR using TaqMan probes. I did relative quantification and so I didnt do a standard curve.. So for quantification I try to use the ΔΔCt method (comparative CT method). The real time PCR report gives me the ΔΔCt values for each of my sample and then I calculate the 2^-ΔΔCT that can be used for my graph. Although, when I do the ΔCt and ΔΔCt calculations according to the manifacturer's instructions by hand I have different 2^-ΔΔCT values and hence different expression profiles for my samples. Could someone gime me advice on that?
Also, what other methods do you use for relative quantification? Which one better in my case.

Thank you very much

Edited by George250, 07 July 2009 - 04:06 PM.

[ivanbio]

Hi George250,

To answer your question it would be best if you posted your data so that we can determine where the discrepancy lies. If you post the Ct values of your assays and the ddCt obtained from the instrument it should not be that hard to determine what the problem is.

[George250]

[quote name='ivanbio' date='Jul 8 2009, 04:16 PM' post='28660']
Hi George250,


Thank you very much !!
Please find attached

Attached Files

•  my_data_from_3_samples.doc   26.5K   105 downloads

Edited by George250, 08 July 2009 - 10:37 AM.

[ivanbio]

After going through your data, the ddCt values I got for samples 2 and 3 were very similar to the ones obtained by the instrument. Mine were -6.06 and -5.9, while the instrument's were -5.9 and -5.92, respectively. This small difference could be due to the way Excel performs these calculations (carries more decimal points, etc).
As for sample 1 I got -5.08 while the instrument got -4.77. That is a larger difference and I am not sure how to explain that one. Yet, even this larger difference between ddCt values implies a difference of 34-fold (ddCt of -5.08) versus 27-fold (ddCt of -4.77). For all intended purposes a big fold difference, regardless of wether it is 34 or 27-fold. My best guess is that this difference comes from the way the analysis is setup on the instrument. There is likely a difference in the way your data is being normalized or something along those lines.