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-   -   Why are some replicates positive and negative? (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/56358-why-some-replicates-positive-negative.html)

lucia.pagani 02-12-2009 04:55 PM

Why are some replicates positive and negative?
 
Can someone explain me how it is possible that for some samples I have one positive and one negative replicate?
For example, during my last RT-PCR I loaded 8 ul/well of cDNA diluted 1:2 for MT1, and I used as housekeeping gene CDK4, loading 2ul/well of cDNA diluted 1:2. cDNA samples for the 2 genes were from the same mix. I have done 2 replicates for every gene.
The results of my RT-PCR are that CDK4 gives great replicates, and for some samples of MT1 I have one positive and one negativer replicate.
Moreover, the Ct of MT1 is quite high (after 34-35 cycles).
I cannot interprete my results, and eather find a solution to this strange problem...
Are you awared of a technique to "decrease" the Ct of a gene?

Aga 02-12-2009 05:23 PM

Re: Why are some replicates positive and negative?
 
Have you ever tried to do this assay before or was it for the first time? If you did, did you have similar results?

One explanation - your housekeeping gene competes with MT1 amplification.
Another thing - you have very little MT1, nearly to detection limit, so you could have amplification in one well and none in the other.

You can get higher Ct if you have more gene in the sample.

Have you tried to amplify it without housekeeping gene to see if they compete with each other?

lucia.pagani 02-13-2009 11:07 AM

Re: Why are some replicates positive and negative?
 
Thank you for your advices. Actually I am doing singleplex, so I don't think that there is a competition between MT1 and CDK4. But I have also thought that maybe my levels of MT1 are near the detection limit; that's why I loaded 4 ul of cDNA/well (I normally load 1). I have done my first RT-PCRs for MT1 with 1 ul/well, and I had similar results. I did a try with 3 and 6 ul/well, and I saw that between 3 and 6 the Ct were not that different, so I chosen 4ul/well. Do you kow if increasing the cDNA/well is the only thing that I can do? I don't think that changing promers/probe amount would modify the Ct, and eather the annealing/amplification temperature, and I don't know what else I could do...

Aga 02-25-2009 11:11 PM

Re: Why are some replicates positive and negative?
 
I know you're doing RT-PCR, but I've recently read this article:
LightCycler qPCR optimisation for low copy number target DNA by I. A. Teo, J. W. Choi, J. Morlese, G. Taylor and S. Shaunak, [Only registered and activated users can see links. Click Here To Register...] (Let me know if you don't have full access and you want to read it)

They deal with qPCR for amplification of low copy of target DNA proposing to run cycle-limited nested PCR using a combination of conventional PCR and real-time PCR. The basic idea is that they first run nested PCR using a small number of cycles and large load of DNA (500ng I think).

They first tested how many cycles are necessary for nested PCR so that the reaction would not reach plateau. Then they transferred 2ul of that product and run real-time PCR (they used SYBR Green, but I think this can be applied to any system including probes). As far I remember they proposed a method of quantification using this approach.

lucia.pagani 03-11-2009 02:18 PM

Re: Why are some replicates positive and negative?
 
Thank you for the advise! I am now doing the limited nested PCR, and soon I'll test the PCR products in RT-PCR.
I hope it will work!
Lucia

Aga 03-11-2009 11:27 PM

Re: Why are some replicates positive and negative?
 
I wish you good luck and hope you'll succeed :)


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