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Something Goofy in My Real Time Results

RT gene knockdown shrna

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#1 Nabin

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Posted 01 August 2012 - 12:45 PM

Hi,
I am trying to establish a stable cell line for IMCD3 cells. I have transfected the cells with fugene for the purpose of gene knockdown. I picked the clones, and did RT PCR for initial screening of the clones. For some of the clones, the quantitative comparision ( 2^-ddCT) is actually showing an upregulation of gene than downregulation. I used actin as reference gene and I had the regular IMCD3 cells as control samples. I have gene knockdown upto 30 % for some clones, but for few clones its like four folds upregulated in transfected cells. I don't know whats going on... Help me.

Nabin

#2 Trof

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Posted 01 August 2012 - 01:34 PM

Did you transfected also with a "mock" vector? Did you measured those cells too?

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#3 Nabin

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Posted 22 August 2012 - 01:31 PM

No I didn't.

#4 luckygiirl

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Posted 24 September 2012 - 08:10 AM

Hey did you ever find out what was causing this problem because Im experiencing the same issues with my qPCR !!





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