qPCR - same Ct for all samples and STD curve

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lavila
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qPCR - same Ct for all samples and STD curve

Post by lavila » Wed May 17, 2006 10:00 pm

Hi everyone,

I have been having this problem for over 3 months now and I'm not sure what else I can do to solve it.

I'm doing absolute quantification of various genes in trophoblast development and I've been using GUS as my endogenous control. The assay has worked before and I thought it optimized but lately I've been having all my samples, including standard curve, coming up around the cycle (giving a Ct between 9-10). The gene of interest works ok (STD curve slope= -2.34 and R2= 0.96) and I ordered new primer/probe thinking it might have been contamination....
I'm out of ideas...Does anyone have a similar experience?

Any ideas are welcome!!

Thanks for helping! :)

Luana

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LilKim
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Post by LilKim » Thu May 18, 2006 3:26 pm

Something similar hapened to me... are you sure that your cDNA is ok?

In my situation it was getting really inconsistent results (partly because I was learning.. and doing my first trials) AND shortly after I scrapped my project the qPCR machine was serviced and they discovered that several wells were not cycling propperly.

if possible, try to recheck your reagents.. and see if you can find a control sample to run (for both your experimental cDNA and your "housekeeping" gene) on the qPCR machine... and then check that the cT values or correct (for the control).

therefore, if the control is working ok... then you'll definitely know that something is wrong with your prep (which kinda sucks ... but at least it gives you a starting point to correct your problem)

I hope you're not too frustrated .. cuz i KNOW how it feels!

Good luck!
- KIM

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