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#1 Sumpf

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Posted 19 March 2009 - 05:11 AM

Hi,

I treated cells with cyclohexamide as well as with my drug of choice --> Drug-/CHX-; Drug+/CHX-; Drug-/CHX+, Drug+/CHX+
I did TaqMan with three different endogenous controls. Which analysis should I do?

Thx in advance.

#2 lotus

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Posted 19 March 2009 - 01:20 PM

what do you mean? could you elaborate on your question?

#3 bob1

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Posted 19 March 2009 - 04:11 PM

Are you aware of what cycloheximide does to cells? PCR isn't the right assay for this experiment.

#4 Sumpf

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Posted 20 March 2009 - 05:13 AM

Are you aware of what cycloheximide does to cells? PCR isn't the right assay for this experiment.


Cycloheximide inhibits the protein synthesis. As I have a downregulation of my gene of intrest I would like to know how this happens. For this I treated my cells with CHX and investigated the mRNA level by TaqMan.

what do you mean? could you elaborate on your question?

Well, I have plenty of results but I don't know how to analyze them. I normaly analyse my TaqMan results with the ddCT method, as I did here too, but how can I see whether my results are significant? I did the experiment three times. The results look similar, but as I said I have no idea how to do the statisical analysis.

#5 bob1

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Posted 25 March 2009 - 04:01 PM

Sorry for the late reply.

Regards the analysis, you will need to do some sort of test, such as chi-square that will tell you the which points are different from each-other. If you have more than 2 sets of points, you will need to do an ANOVA or similar test, which can be followed by a Tukey's multiple comparison test to distinguish which component(s) are the significantly different one(s). These should only be done after an ANOVA or similar test, as the risk of a type one error is significant if used on their own. Have a look at the Wikipedia page on multiple comparisons, it seems to be OK to me.

There should be tests available for testing between curves if you have generated quantitative curves, but i have never used them, so I can't give you much advice other than to plot the 95% confidence intervals, and see if they overlap.
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So the chx inhibits ALL protein synthesis - what does this tell you about mRNA or the protein state of the cell and the interaction between the two? As far as I can tell, next to nothing. You could just have treated cells with your drug, taken a western lysate and examined that for candidate proteins. As the chx is inhibiting synthesis of proteins, it is also inhibiting synthesis of RNA and DNA polymerases, helicases etc, how can you tell what is the drug and what is cycloheximide with such a large effect on the cell as it is, despite the various permutations of drug and chx you have, as the drug will be affecting things the the chx is also.

mRNA levels do not equal protein levels in the cell. It is a common fallacy to believe that they are equivalent, but there is quite a bit of evidence of some mRNAs being stored for later translation.

#6 leelee

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Posted 25 March 2009 - 07:32 PM

bob1, that is an interesting point you make about mRNA being stored for later use, could you possibly give me some references regarding this, I would very much like to learn a bit more about it :P

Cheers

#7 bob1

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Posted 26 March 2009 - 05:04 PM

Couple of refs below:

Science 21 October 2005:
Vol. 310. no. 5747, pp. 486 - 489
DOI: 10.1126/science.1115791

J Bacteriol. 1978 October; 136(1): 2434.




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