Hello Edna Wanjiru

Do you have a reference dye added to the PCR mix? Usually, a reference dye is used in qPCR reactions to normalize for non-PCR related fluorescence signal variation.

Best Wishes.

Hi,

I would suggest to optimize your PCR assay first on a regular PCR instrument, and conditions and see the results on an agarose gel. I guess you may have done this already. If not, optimize first and then move tot he qPCR instrument to avoid struggling with the unexpected results

Hai Edna Wanjiru

In my opinion, you need to check whether sample that you used to perform standard deviation expressing this gene. You can check the CT value of 100 ng of your sample. If the CT is above 29 then you need to change to the sample that expressed the gene.

Secondly you need to optimize the annealing temperature of the primer by doing gradient qPCR.

Hope this will help.