elp with qPCR standard curve - (Dec/06/2007 )

Hello

I am trying to detect the gDNA copy number of virus infected cells.
I want to use a gDNA to make a standard curve for my real samples.
I know my gDNA is 64700 copies/50ng gDNA.
I make serial 2-fold dillutions of that gDNA. (50ng, 25ng, 12.5ng .....)
Then run normal PCR for these gDNA dilution serials by using PFU enzyme.
After this first PCR, I dillute the production 200 times and then take 3ul of each sample to run qPCR by using iQ SYBR supermix on Bio-Rad machine. I did got a linear line on these samples. But the Ct is between 19 -22 (total 8 dillutions). And the efficiency is 400%.
What is the reason for this problem? I got this protcol from other lab, they use the same primers and same protocol and same gDNA samples. Only thing is that they are runing on AB machime and also use the AB reagent.

Also, I found that my maxium Ct value always is around 23. I cann't get Ct higher than that. What is the problem for this?
It is because of the Bio-rad machine or others??

Thanks

Can you give some more details about this section? it`s not quite clear for me. you are doing qPCR with standard curve PCR endproducts of a conventional PCR?

Thanks for your reply.
Originally, I use a plasmid as the standard curve to detect the copy number of the gDNA. There are two problems: 1. the plasmid copy cann't lower 2000, otherwise the siginal will be not in the linear range. 2. I start from 300ng gDNA to run qPCR and the siginal looks like background. I don't know how could you detect only 100 - 10 copies in the starting gDNA samples?
That is why we try to follow another protocol. use a gDNA(I know how many copies/50ng gDNA) as standard. When I am doing the experiment, I make serial dilluitions of the standard gDNA. Then run these gDNA by normal PCR, same operation for my samples. After the normal PCR, I take same volumn of the gDNA standard and also my samples to run the qPCR. Here, the idea is to appplify the copy by normal PCR for both standard and samples. I did same operation on the standard dillutions and my samples.

My gDNA sample is coming from the virus infected cells. Seems I cann't directly detect the copy from the gDNA sample. I have to run normal PCR to amplify the copy and then run qPCR to detect the signal.

Another problem here is: I got 300% efficiency on my gDNA standard curve. What is the possible reasons for this?

Thanks

if i understood you correct you are doing some kind of preamplification using normal pcr. the problem is, that this preamp step is not quantitatively. that means, the ratio of target template between 50ng, 25ng, etc... dilution points after the normal pcr will not reflect the original amount. i think this is one cause for the weird efficiency values.

what is the efficiency of your plasmid standard curve?

and what is your experimental approach? you infect cells with a virus, then isolate gDNA from the whole and try to quantify the viral gDNA copies within the cells?

Yes. You are right. I am trying to detect the virus copy in the infected cells.
For the plasmid standard curve, I can get reasonable efficiency. The linear range is from 60million copies to 5000 copies. But there is another problem, why I cann't continue lower the copy number to 500 or 50. I heard that some some can detect single copy.

I use this two step PCR just because I cann't detect out the copy number signal directly from the gDNA material. I need amplify the copy first, then try to get the signal. I got this protocol from another lab and they can do it. Only difference is I am using Bio-rad machine and they are using AB machine. (all are SYBR reagent.)

One more problem. When I use the water as the negative control, the Ct value is around 25. I expect that the Ct should over 30 at least. And I checked the melting curve, single peak around 84 degree. What is the possible reason?

Thanks

concerning the positive water controls: this can be easily due to contamination. you are subjecting PCR endproducts (have you checked them on a gel?) which are present in really huge amount to subsequent PCR analysis. Use filter tips and keep opened post-PCR vials away from the place where you are preparing your PCR mastermixes etc.....

Maybe its also worth trying to dilute the template much more before the second amplification step. I have done already standard curves with RT-PCR products and I started with a 10^-5 dilution which gave Cts about 15 down to 10^-11 (Ct ~35). You should also purify your PCR products before the second step (but if this works quantitatively?).

and you should also investigate the cause for the low sensitivity. maybe bad primers, bad conditions or both.