I have designed a set of primer from human gene within an exon. I used this set of primers to amplify in cDNA from human blood and used UHRR as a positive control, but it did not work. however, when I used human gDNA from blood, it works. I did sequencing for the PCR product, and it confirmed the sequence is the one I am expected. from the result, I am sure the primers are working, and the PCR protocol has been optmised. The question is why it does not work in human cDNA sample and UHRR?
I was thinking if the gene has low expression level in human blood, however, when I used the other primer set, it works perfectly.
Thanks for your time to read the question. any comment will be appreciated.