high Ct and differents Ct for the same template - (Oct/26/2005 )

Hello,

I’m trying to analyze my samples by LightCycler. What happens is that my gene of interest is expressed at low rate and the templates have a high Ct so the efficiency of reaction decrease at this level. I think is due to the worse reaction efficiency at high cycles that when a run a serial dilution of template to evaluate the reaction efficiency the duplicates not to duplicate well and sometimes the same sample has different Ct. The dilutions that I use are: ½, ¼, 1/8, 1/10, 1/12 or 1/5, 1/10, 1/20, 1/50, 1/100, 1/160.

My primers are specific I only have my product, I don’t have contamination and not primer-dimers problems. I think is not pipetting error because I don’t have problem with other genes. And I can’t use more starting material.

Could you give me some suggestion? Thanks a lot.

-pilar-

QUOTE (pilar @ Oct 26 2005, 09:05 AM)

my gene of interest is expressed at low rate and the templates have a high Ct so the efficiency of reaction decrease at this level. I think is due to the worse reaction efficiency at high cycles that when a run a serial dilution of template to evaluate the reaction efficiency the duplicates not to duplicate well and sometimes the same sample has different Ct.

I'm not familiar with the Lightcycler, but have you done a regular RT-PCR on your sample? If so, at what cycle does your target appear on the gel? If you aren't willing to add more sample, have you tried modifying primer/probe/Mg conc?

-Orion_XR-