Different dilution factors

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Different dilution factors

Postby lina86 » Thu Apr 05, 2012 8:19 am

Hi!

I am using a relative standard curve method for my gene expression assay and I am struggling to get good curves. This might be a stupid question, but I am wondering is it possible to make different dilutions for the gene of interest and endogenous control standards curves? For instance the gene of interest isn't expressed very well so I would need to use dilution factor 1:2 to get a proper curve. However, endogenous control is expressed normally and dilution factor 1:10 would be okay. Or should I use the same dilution factor for both of them (something in between)? I am using TaqMan chemistry.

Thanks in advance! :)
lina86
 
Posts: 1
Joined: Thu Apr 05, 2012 7:39 am

Re: Different dilution factors

Postby Mikael Kubista » Thu Apr 05, 2012 2:53 pm

If you use small steps in the serial dilution the estimated efficiency will be uncertain. Make sure to always calculated the confidence interval for the estimated Efficiency so you know its reliable! If you use GenEx, its done automatically!

What is the purpose of your standard curves? If the object is to validate the performance of an assay, you can use cDNA from a library. Several cDNA libraries are available, for example, here: http://www.tataa.com/products/cDNA/View ... ducts.html.

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


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