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microarray after overexpression and silencing

microarray

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#1 Fahad

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Posted 18 October 2011 - 05:03 AM

Hello everyone,

I have a questions that I need your kind help to answer it.

My experiment:

I've overexpressed a gene of my interest in cells that do not normally express it. For this, I used a vector cloned with this gene which was amplified in E.coli before being purified by Qiagen Maxiprep. My negative control was an empty vector amplified and purified in the same way.

Then I also managed to silence that gene in other cells that normally express it abundauntly using siRNA. My negative control was an unspecific universal negative siRNA control.

In both experiment, RNA was extracted for microarray analysis to explore which genes have been affected by the overexpression and the silencing.


My questions:

In case of the overexpression experiments, I noticed that the viability of cells transfected with the empty vector negative control was always reduced suggesting the presence of bacterial DNA contaminants. So, how can I exclude the unspecific effects of the bacterial DNA contaminants that might eventually affect the microarray results?

Thank you in advance.

#2 FishDoctor

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Posted 19 October 2011 - 09:03 AM

The effects on the cells from the transfection of the plasmid dna are going to be there, and that is why you have that as a control. These effects are seen all the time when cells are perturbed by transfection. I dont think you have anything unusual going on, the dna is fine. Do your array and look for changes as compared to the empty vector control.

#3 Fahad

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Posted 20 October 2011 - 01:02 AM

Hi FishDoctor,

Thank you for your reply.

Yes, the array comparison will be between the +ve and -ve vector effects on one side then between the overexpression and the silencing on the other side. I'm just afraid that the -ve control will drive unspecific alterations in genes expression which will have an impact on the comparison after all! Do you recommend some vector that is known to have the minimal effect on the transfected cells?

Edited by Fahad, 20 October 2011 - 01:02 AM.






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