qPCR Error bars problem!

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qPCR Error bars problem!

Postby toffee89 » Wed Nov 26, 2014 4:41 pm

Hi
I need help with some qRT-PCR error bar calculations. I have carried out all of my qPCR with my gene of interest and of my housekeeping gene and have calculated my fold-changes for my gene of interest with the delta-delta-Ct calculation. However putting error bars on my graph showing the fold-changes is confusing me. What data should I be using to calculate this? I can't use the normalized Ct values as this is not correct for fold-changes and when I convert my Ct values (for my calibrator and experimental) using the delta-delta-Ct calculation and then get the SE for that my error bars are bigger than my fold-change. Can anybody shed some light on this and where I am going wrong? I can't use the software that comes with the qPCR machine as it is getting confused with how my plates are arranged.

Thank you in advance!
toffee89
 
Posts: 2
Joined: Wed Nov 26, 2014 4:11 pm

Re: qPCR Error bars problem!

Postby Mikael Kubista » Fri Nov 28, 2014 8:10 am

It is tricky to do these calculations right! See step by step instructions in the tutorial:

. Kubista, R. Sindelka, A. Tichopad, A. Bergkvist, D. Lindh, A. Forootan.
The Prime Technique. Real-time PCR data analysis.
G.I.T. Laboratory Journal (2007), 9-10, 33-35.

(available on www.tataa.com) if you don't have access.

What instrument do you use? All leading instrument companies collaborate on this matter on GenEx, which is a sofware to merge independent runs into a single analysis and do all kinds of statistical comparisons. It also calculates the error bars correctly.

http://www.tataa.com/products-page/qpcr_data_analysis/

Good luck!
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm

Re: qPCR Error bars problem!

Postby toffee89 » Mon Dec 01, 2014 1:27 pm

Hello,

I'll have a look at that publication and have another try. I am using the Stratagene Mx3005 qPCR machine but the software gets confused by my setup. I will also look into Gene Ex software.

Thank you for your reply and help - much appreciated.
toffee89
 
Posts: 2
Joined: Wed Nov 26, 2014 4:11 pm


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