Different Runs Of Real Time Pcr, Comparing Expression Of All Genes According To Housekeeping Gene

Question

[Biomonika (Noolean)]

Hi all!

I am doing some real time pcrs, at each run with set of genes of interest and one housekeeping gene.

I would like to compare results of all this runs, so I could know relative expression levels of all my genes.

And thats when the problem comes. I have read articles about ∆∆CT method, which, according to my knowledge, compares genes of interests and housekeeping genes in both treated and untreated tissue.

I guess that my case is different - I have only one tissue (untreated) and I want to compare expressions of many genes, which unfortunately forces me to do many runs.

How can I do such comparision? We lead passionate discussions in our lab, so I will be really thankfull to hear your opininon! Thanks a lot :-)

[Damian Kao]

So you are trying to look at the relative expression of genes within one sample? How many replicates did you do?

[Biomonika (Noolean)]

For example in first run I have 3 samples of leaf of plant number 1, 3 samples of root of plant number 1, 3 samples of leaf of plant number 2 and so on, so always 3 samples of different parts of different plants. In next run I have the same categories (leaf, root and so on), but also different plants. In every run I use 3x10 samples, so 3 identical samples of 10 things. And I want to normalize all data I get so that I could say: there is more expression of this gene in root than in the leaf or anything else. I simply want all data from all runs to be comparable:)

Answer 1

[Larry_Parnell]

Although performed with mouse, you may be interested to learn that many so-called "housekeeping" genes are not expressed in some tissues. Over 1000 housekeeping genes were found not to be expressed in one of a panel of mouse tissues. This was done by examining expression in many tissues and observing expression in all but one (Thorrez, Schuit 2010 Genome Res). Thus, your choice of reference "housekeeping" gene is critical.

[Biomonika (Noolean)]

Thank you, I know that. We did this experiment with more housekeeping genes because of this. When asking my questions I count with unreal assumption of housekeeping genes behaving ideally:) When I uderstand how to interpret my results in this ideal word, then I could proceed with more complex assumptions, which are much closer to real world and real behaviour.

Answer 2

[eszterhegyiova]

Hi Noolean! It was an earlier post, I am trying to do something similar in pancreas tissue samples, but also have problems with data analysis. Have you figured it out how to do the analysis of your samples? thank you very much for your answer!