Ct for housekeeping gene is higher than Ct of target gene. - (Jan/30/2007 )

Hi all,

I am trying to do relative quantification on gene expression and i am using SYBR green dye. However, i found that the Ct value of my housekeeping gene (cyclophilin) is about 32-38, and the Ct of my target gene is about 22-30. Is the Ct value acceptable?

To create a standard curve for the housekeeping gene, after a 5 fold serial dilution, amplification only detected from 3 point of the dilutions.The PCR efficiency was about 91.6%, slope=-3.542. Is this standard curve acceptable? My template is limited, i had tried 2 fold serial dilution, amplification from 4 points were detected but the efficiency is about 300-400%. Any suggestion?

a Ct of 32-38 is not so good. your efficiency is nice ,though...it's up to you, but I would find an alternate housekeeper, or try alternate primer sets for the same housekeeper, in an effort to lower the Ct to a more believable range. if it's in the 32-38 area, that's a pretty tiny amount of template - this means your results will not always be consistent; there will be more error and higher std dev