copy number and sampling. - (Dec/13/2007 )

Does anyone imagine the following scenerio. We have a result we are trying to explain. We are looking for tumor cell presence in peripheral blood of cancer patients. We are using RT-PCR and primers for CEA. All controls are beautiful. Lines that express show up, control normal donor PBMC are flat negative. In the patient samples, we draw 5microliters from say a 50ul stock of RNA to generate the DNA before PCR. Of 30 patients, in one run, a few come up positive. We repeat and a second group come up positive. Is it a plausible explanation that copy number is so low that when you sample you can get +s one time and -s the next from the same sample? Am I dreaming or is this possible? If sampling were the case, how many times would you run the reaction on 30 samples to insure you have gottent the correct number of +s. Suffice it to say that PBMC from normal donors run multiple times do not come up positive., It is only patient samples where I expect some to be +. This is not the case with all antigens. Some can be repeated multiple times and the same samples come up +. I assume the copy number is at a point where it indeed is evenly distributed in my sample. Is this possible or is there a more logical explanation I am not thinking of? Thank you

sometimes PCRs just fail. have you tried running the samples in duplicate or triplicate?

V