5 replies to this topic

[Bunsen Honeydew]

Hi,

I'd like to analyse my data using the 2-ΔΔCT method, but I'm a little confused as to the Ct value I should enter into my excel spreadsheet when my samples do not amplify.

I'm working with the following cell types/conditions, looking at the gene expression of lots of other CD molecules :
[control]     1) CD8+ CD56- cells
[case]          2) CD8+ CD56+ cells
[case]          3) CD8+ CD56- cells which have been chemically stimulated
[case]          4) CD8+ CD56+ cells which have been chemically stimulated

Ideally, I would like to compare 2, 3 and 4 to my control sample, number 1.  My HK gene amplifies perfectly in all cell types/conditions, each producing consistent & reliable Ct values.  My problem is with my GOI - depending on which gene I am looking at I can see up/downregulation or no expression at all.  No expression is quite common in samples 1 & 2, which then means I have no Ct value to enter into my spreadsheet (which I of course need, so I can compare all my case samples).  What do people do in this situation?  Would you enter a Ct value of 0, or enter the value of the total number of cycles you have run?  Or should I change my control to another sample, say 3 or 4, which regularly amplify?

Any help and suggestions would be greatly appreciated.  I'm getting some weird results (eg, 159146-fold upregulation - I'd expect upregulation, but this much is insane!) which I think cannot be right.

Thanks in advance,
Bunsen

Attached Files

•  test.xls   38.5K   98 downloads

[ElHo]

If no amplification can be detected, we usually enter the total number of pcr cycles.

[Bunsen Honeydew]

ElHo, on 17 August 2010 - 02:12 AM, said:

If no amplification can be detected, we usually enter the total number of pcr cycles.

Thanks ElHo - I'm the only real-time PCR person in my lab, so have no one to ask these silly questions  

[fishdoc]

Bunsen Honeydew, on 23 August 2010 - 03:07 AM, said:

ElHo, on 17 August 2010 - 02:12 AM, said:

If no amplification can be detected, we usually enter the total number of pcr cycles.

Thanks ElHo - I'm the only real-time PCR person in my lab, so have no one to ask these silly questions  

I had this situation in my dissertation. I just put in "40" for those values, and a professor on my committee instructed me that is the wrong way to go about it. By giving it a value of "40", you're assigning false data. You're assigning a value with no evidence of that being the real value. He told me that anything that is undetectable should be labeled as such - undetectable or some other notation that explains the amplicon was not detected.

[NicoBxl]

I've a similar problem

I want to analyze two samples A and B.

For some genes, I've a CT of 40(so undetected) in A and 30 in B.

How can I calculate a foldchange for this ?

If it's impossible how can I present the data

Thanks

[Trof]

2-ΔΔCT method tells you how many times is your sample down/upregulated in comparison to your control. If you don't detect any amplification, you should just state so as fishdoc said. You just say that sample is downregulated to undetectable level. You can't say how many times zero is lower than anything, that doesn't make sense.

If you have no detectable amplification in your control (calibrator), then you should choose another one that amplifies well.

Edited by Trof, 01 February 2011 - 04:03 AM.