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Too high between-run / between-sample variation in real-time PCR experiments?

Too high between-run / between-sample variation in real-time PCR experiments? - Protocols and Methods Forum

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  #1  
Old 09-25-2003, 04:25 PM
Paul Wary
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Default Too high between-run / between-sample variation in real-time PCR experiments?



What are factors increasing between-run or between-sample variation in
real-time PCR experiments and how can I minimize them?

I have only little variation in replicate measurements with cDNAs from
one tube but I have too high a variation in measurements with cDNAs
coming from different tubes but still from the same source. I had
several cell culture flasks with the same cells in them. I isolated
total RNA from each each cell culture flask separately and performed
reverse transcriptions for each RNA in separate tubes. But since the
RNA is from the same cell type, just from different flasks, I should
essentially get the same results. But the Ct (threshold cycle)
differences go up to 1.6, which considerably affects the results
because of the exponential nature of the formula used for evaluation
(2 to the power of minus delta-delta Ct).
May there have been signficant differences in the efficiencies of the
reverse transcriptions alhtough they were performed in parallel and
simultaneously? If yes, what can I do about this?

Paul
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  #2  
Old 09-26-2003, 08:43 AM
Duncan Clark
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Default Too high between-run / between-sample variation in real-time PCR experiments?

Historians believe that in newspost
<[Only registered users see links. ] > on Thu, 25 Sep 2003,
Paul Wary <[Only registered users see links. ]> penned the following literary
masterpiece:

But only if you have the identical amount of RNA in the starting sample.
You may think it should be the same but how did you check that it was.
If the RT step is identical then the variation is the starting material.
How did you normalise the RNA before you put it into the RT tubes.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 09-26-2003, 02:32 PM
Peter Frank
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Default Too high between-run / between-sample variation in real-time PCR experiments?

Duncan Clark <junk@[127.0.0.1]> wrote:

Funny! :-)


I assume I used the identical amount of RNA in the starting sample
based on my spectrophotometrical measurements.

I determined the concentration of the RNA solutions by measuring the
absorption at 260 nm (and 280 nm to determine purity). Then, I
calculated the volumes I had to add to each reverse transcription
reaction tube in order to get equal (ug) amounts of RNA.

How reliable is this method? Do you think this is where my variations
might come from? If yes, what I can I about it?

Peter
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betweenrun , betweensample , experiments , high , pcr , realtime , variation


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