Dear all,

I have been doing qPCR (after reverse transcription of my RNAs) some time now. I am using sybr method. Ive tried 8 cDNA. Initial cDNA amount is not equal in each sample. Because my RNAs are very low therefore during cDNA conversion all 15ul comes from my samples. And also I am using actin as internal control thus I thought that delta delta ct calculation will eliminate initial unequal cDNA amount (maybe this idea is all wrong please let me know if it is). Nevertheless, with actin primer, some samples give nonspecific amplification therefore their ct values are higher. I understand this from melting curve, you may see attached picture. But my test primer which belongs to another gene of interest did not give such problem.

I do not understand what is my problem?

My knowledge on qPCR is limited and I am eager to learn. Please let me know what should I do? Thanks in advance.

Sincerely,

Suleyman

More Suleyman Bozkurt's questions See All
Similar questions and discussions