How to know tissue specific gene expression by real time PCR - (Nov/17/2007 )
I have been doing real time PCR using Biorad iCycler machine and SYBR green for a while. My interest have always been to compare the expression between two samples, say infected vs uninfected. I did not have any problem, as I used delta delta CT methods.
Now, the problem I have is, I want to see the expression of a specific gene in different tissues. My question is how do I do it? Since I will have nothing to compare with.....I just want to see is the gene expressed?? If yes, what level?? So, please suggest me the strategy that I should follow.
In my opinion you just have to do what you were doing before. But then you don't normalize for a control/untreated but you can normalize for one of the tissue, let say A. And so, the gene expression in the other tissue would be 1, 2, 0.5 - fold different from tissue B.
Are you normalizing your results with internal controls?!
I am doing as you said. I normalize with UBI3, both infected and uninfected.
At this moment, my interest is to know what is the basal level of expression...Is this gene really expressed in the uninfected tissue? I have seen in this situation people mostly doing norhtern. I was wondering if someone has suggestion on how can I answer the same question without doing northern, say without comparing with any other tissues.
At the moment you are doing relative expression... when i read your post my first tought was "why not absolute quatification?" Have you tought about that... i mean, you would avoid some problems if relative expression...