Inconsistent results from replicates in Taqman real-time PCR - (Jun/24/2006 )

Hello all,

I am doing a Taqman real time PCR to detect and quantify bacteria DNA in human blood samples. My problem is that I run each human sample in three replicates but I don´t get the same value in the replicates. It seems that there´s some inhibition but I really don´t know what can I do.
I,ve thought about adding some adyuvants to the PCR reaction mix. Can anyone recommend me some?


Good luck with your experiments!!

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do you make one tube and split it 3 ways to get your replicates? if so, you have a real problem somewhere in your system if you do not get similar numbers...perhaps have your machine serviced first and then check through your components for problems

or, do you mean there are 3 similarly-treated samples that fail to correlate?

at any rate, I would not try to 'fix' the Ct's with adjuvants; if it actually works it will only mask your real data, not fix the underlying problem

what are your efficiencies? are you using assays-on-demand, or self-designed primers and protocol? what have you done to control/test for the quality of your RNA samples?

if you can give some more information we may be able to pinpoint the problem...and when you say the answers are off, how far off?

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Vortex your samples before you pipet the amount you use for real-time and remember to spin your plate

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Hello hobbes,

First,thank you very much for your reply.

I really have a problem because I meant that I make one tube and split it 3 ways to get my replicates

I am using my self-designed primers and protocol to amplify bacteria DNA, no RNA. It is very specific because when I run the product of the capillaries where I have got a signal into an agarose gel I just see one band corresponding with the bacteria amplicon. It seems that the human genomic DNA interference with the bacteria DNA or something related with the DNA extraction procedure... Oh.....it´s quite fustrating!!

Another thing I have seen is that when I perform the real time PCR the same day I have isolated the DNA (with Mobio kit) I get no signal!!If I repeat the PCR with the same eluted DNA after two days at 4ºC some replicates start giving signal(???).

I use the LightCycler MasterPlus Hybprobes (Roche). I can give you more detalis if you want.

See you!

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Hello Dnafactory,


I always do it the way you recommend but still doesn´t work.I don´t know why...I am getting mad because of it!

Thank you very much for your answer!!!

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madeinspain.
What are your typical Ct values? If they are above 35 cycles some variation is to be expected because of statistical reasons (Poisson distribution).
Serge

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Hello Serge,

Yes.All my Ct values are above 35. Then, what should I do?I am quite lost...


Thank you very much for your answer!!

Maria

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we also look at some genes that have CT values above 35, and the error is so great its frustrating. We have tried to increase RNA input into the cDNA mix, but basically if its a such low abundance there really isn't much you can do, sorry...We decided to ditch the ones that gave us inconsistant data, and try other genes that would hopefully tell us the same answer to our question and amplified better in realtime.

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I have another question, regarding replicates. What variation between Ct values of replicates is allowed? So far I only used the Ct values of my replicates if they don't differ more than 0.25. Is this correct?

Many thanks. Yvonne

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Are you sure you don't have proteins or salt or whatever can inhibit the rezction? What do you use to isolate the DNA?

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