qPCR copy number analysis

5 posts / 0 new
Last post
kellie
kellie's picture
qPCR copy number analysis

Hi all
I am trying to determine DNA copy number in patient peripheral blood after infusion of transduced cells. I am getting a lot of background in early cycles and in negative samples, I have tried to set the baseline higher and played with the threshold but it doesn't seem to help and I am getting CTs of 2 which is not right!! I am using Taqman, an ABI Step One plus machine and multipex VIC/FAM probes quenched with TAMRA. The samples are lysed in SDS/PK from frozen cells.

Ivan Delgado
Ivan Delgado's picture
Hi Kellie,

Hi Kellie,

When you say you've tried to set the baseline higher, how high have you gone? If I recall the StepOne has a default baseline of 3 to 15 PCR cycles. If you are getting signal before cycle 15, then that could be explaining your Ct values of 2. If you have not tried to go as low as a baseline of 3 to 8 PCR cycles, give that a try. The important thing is never to have a baseline that overlaps with the earliest signal of your PCR reaction. In other words, if you can see signal at PCR cycle 10, then your baseline should be no higher than cycle 8.

Another question: did you validate your assays as separate qPCR reactions before multiplexing? You need to make sure that both assays give you an efficiency of at least 95% before multiplexing them. On top of that, once you've validated the assays separately, you need to validate them multiplexed to make sure the assays are not affecting each other.

Good luck

kellie
kellie's picture
Thanks, I will definitely go

Thanks, I will definitely go back and do some more quality control. With the low baseline, are you talking about the minimum or maximum?

Ivan Delgado
Ivan Delgado's picture
You are welcome. Yes, a low

You are welcome. Yes, a low baseline implies a low range, like cycles 3 through 8. The important thing is that the upper part of the baseline range be lower than the first signal detected by your assay. If your most sensitive assay starts showing up at PCR cycle 10, then your baseline should be lower than 10, like 3 through 8. A good rule of thumb is to have a baseline that is at least a couple of PCR cycles below your most sensitive PCR assay.

kellie
kellie's picture
Hi Ivan,

Hi Ivan,
I did these tests and the efficiencies are fine, however separating the primers has exposed a bigger problem! I am using a primer/probe set labelled with FAM-TAMRA to the light chain of the single chain receptor I am transducing and a primber/probe set labelled with VIC-TAMRA to a genomic control. When I use the genomic primer/probe set everything is as expected. However, when I use the primer/FAM-probe set to the construct I am also seeing a signal in the VIC channel. It is nearly one log lower than the specific signal but gives nice looking curves and a standard curve of the expected slope/efficiency. This happens if I use transduced cells or plasmid alone, but not in the water control. Note there is no genomic primers or VIC probe present in this reaction!! I digested some of the probes with DNase1 and got negative signal from FAM probe in the VIC channel.
Has anyone seen anything like this? Any ideas??