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