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Go Back to: GenRep's Corner
Scientific Question

Genomic Repairman
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Scientific Question
Thu, Oct 14, 2010, 1:03 pm CDT

Hey guys, I have an issue with something I'm doing and wanted to bounce an idea or two off of you.  So I recently created stable knockdown cells lines for my gene of interest using four different targeted shRNA constructs.  One of my cells actually shows an increase in both RNA and protein levels (WTF, I know).  I'm guessing maybe my shRNA is possibly affecting a miRNA that is regulating my gene, I blasted the shRNA sequence and its pretty specific to my gene so I don't know what the heck is going on.  Ideas?


Nikkilina
Washington University School of Medicine
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Thu, Oct 14, 2010, 9:44 am CDT

Wow! I've never had that problem. I'll ask a couple of people in my lab who have more experience than I do with it. I'm still trying to get my knockdowns to work at all!


Genomic Repairman
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Thu, Oct 14, 2010, 9:46 am CDT

Yeah, I know crazy right?  Are you doing si or shRNA knockdown?


Nikkilina
Washington University School of Medicine
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Thu, Oct 14, 2010, 10:10 am CDT

Short hairpin. I'm getting knockdown, but I'm having issues with my MOIs because there are only crappy antibodies for my protein, so I have to load 70ug onto a Western. Unfortunately, astrocytes don't have a lot of protein, so it just complicates everything!


Brian Krueger, PhD
Columbia University Medical Center
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Thu, Oct 14, 2010, 10:49 am CDT

Start over, you screwed something up.


Genomic Repairman
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Thu, Oct 14, 2010, 10:57 am CDT

Have and its doing about the same thing.  Also construct has RFP so, I know that its in there too.

Nikki as a proof of concept that your shRNA is working you could make or obtain a cell line that overexpresses a tag version of your protein, do your RNAi and measure KD to see how effective your shRNA is?


Nikkilina
Washington University School of Medicine
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Thu, Oct 14, 2010, 1:03 pm CDT

I've already got the proof of concept. It works to knock the protein down, I just need to figure out which dose of virus to give, and that's the problem. I'm hoping to get it done Monday.

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