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Taqman real-time PCR


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#1 ropa

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Posted 13 March 2010 - 09:32 AM

Hello,

I have some general questions that might seem stupid, but I am really confused right now. I got Ct values as low as 10 to about 13 in my NTC and no-RT controls using Taqman. Maybe I am running the controls wrong so I was asking for help from you guys: This is what I had:

The NTC control had water in place of the cDNA and probes.
The No-RT control I am little confused by. What is really suppose to be in a no RT control and is it the same as the NAC?

Thanks

#2 tea-test

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Posted 13 March 2010 - 09:52 AM

hi,

in order to help you with the first problem we would need a picture of the amplification curves. It is very unlikely that this is caused by an actual contamination as the Ct values are very low. have you set the baseline and tresholds manually?

The idea behind the RT- control is, to find out if you are detecting genomic DNA with your assay. In a reaction containing only pure RNA (no reverse transcriptase added) no DNA amplification should occur. If your RNA is contaminated with DNA you might get a signal depending if your primers are exon spanning or not...

what is NAC?
tea-test: The artist formerly known as Ned Land

#3 ropa

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Posted 13 March 2010 - 11:36 AM

I will try to get the amplification curves, I cannot access them right now. I guess the no RT control that I have is okay then--RNA, Taqman Master Mix and probes? NAC is no amplification control, some people describe it as cDNA,probes and no Master Mix, essentially no Taq pol in the reaction mixture and obviously we should not get any amplification.

Edited by ropa, 13 March 2010 - 11:40 AM.


#4 Prep!

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Posted 17 March 2010 - 04:23 AM

I will try to get the amplification curves, I cannot access them right now. I guess the no RT control that I have is okay then--RNA, Taqman Master Mix and probes? NAC is no amplification control, some people describe it as cDNA,probes and no Master Mix, essentially no Taq pol in the reaction mixture and obviously we should not get any amplification.



I wonder why wud u use a Taq-less sample for some kind of a control!!! correct me if i am wrong...
using a no template makes sense... also the RT control makes sense.. but if u wanna chek if there is amplification without the Taq as well as the dye??!!! all i can think of is tat it can be used as a system suitability for ur machine... but then the other two controls shud solve tat purpose..

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#5 vladooo

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Posted 22 March 2010 - 07:41 AM

It could be caused by your probes degrading unspecifically during cycling - either you stored them wrong or are very old. This could be seen at the amplification curve which does not follow typical exponential growth. Also, it should be seen in all reactions, not only NTC.




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