Hello everyone, I have a question that related to ABI Biosystem Stepone for detection whether virus exist or not in my cell culture sample.

I should detect specific virus type in my cell culture sample (cell pellet). I already design for primer forward and reverse, probe that can recognize that virus when I perform PCR. I have virus plasmid as positive control in my experiment.

I have plan to perform real-time PCR experiment with these samples:

- Negative control (NC): Template is DW.

- Positive control (PC): Template is viral plasmid

- Positive control - Sample (PS) (ratio mix 1:1): template is viral plasmid+ cDNA (isolated from my cell culture sample which I want to test)

- Sample (S): template is cDNA (isolated from my cell culture sample which I want to test).

I used ABI Biosystems for my real-time PCR test. It is the first time I perform this experiment so I do not know how can I choose And my question here is:

1. What program I should choose for my experiment? I think I should choose "Comparative Ct Experiment", am I right?

2. I part "Plate setup":

- At Target setup, how many target should I choose?

When I perform gene expression qRT-PCR, I choose 2 target: 1 is my interest gene, and other is GAPDH as endogenous control. But I this kind of experiment I do not know how to choose.

- At Sample, How many samples should I choose?

As I mentioned before that I should running with 4 samples: NC, PC, PS, S.

- At Setup "Relative Quantification setting":

which sample I should choose as reference sample?

which target I should choose as endogenous control?

I hope anyone who know about this experiment can help me. Thank you so much for your help.