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Gene knockdown expression levels shRNAmirs

Gene knockdown

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#1 marspimp

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Posted 17 January 2013 - 04:39 PM

Hi guys,!
I was hoping someone can help me figure something out. I am doing a time course study using 3 shRNAmirs. I am looking at 48 and 72 hour time points. I have done a qPCR to analyze my data and from the results, I see that my gene of interest, the knockdown efficiency is significantly high at the 48 hour time point. But at the 72 hour time point the knockdown efficiency is quite low compared to the 42 hour time point; less than 50% knockdown. Whereas after 48 hours, there is more than 80% knockdown.

I expected the expression level to be even low at 72 hours. I had used the same concentration of cDNA for the qPCR.
,
Any ideas why this might have happened?

#2 pcrman

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Posted 28 January 2013 - 02:48 PM

I am confused by your numbers. you said:
  • the knockdown efficiency is significantly high at the 48 hour time point.
  • But at the 72 hour time point the knockdown efficiency is quite low compared to the 42 hour time point; less than 50% knockdown.
  • Whereas after 48 hours, there is more than 80% knockdown
Can you clarify?

#3 marspimp

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Posted 29 January 2013 - 12:07 PM

So to clarify,
when I collected my cells at the 48 hour time point, extracted the RNA, made cDNA and then did a qPCR. I did the same thing with my cells that I collected after 72 hours. I did the qPCR to see the efficiency of knock down of my gene of interest, at the two time points.
At the 48 hour time point, I saw more than 80% knock down of my gene of interest. Whereas, at the 72 hour time point, I did not see as high a knock down of the gene. It was less than 50%! I was expecting the knock down of my gene to be even higher after 72 hours ...
So, I am wondering what the possible reasons could be? Non-specific knock down? Or, after 72 hours, the cells are far too confluent so not enough knock down ...?





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