Hi,
I've designed qPCR primers to target my gene of interest in HeLa cell line. I designed them such that it will encompass an exon-exon junction and it the product is 100-200bp long. Apparently, the first set of primers did not amplify, so I re-designed new ones until I reached 5 different sets and still, all of them could not amplify anything. I'm starting to think that maybe the gene has very low expression, thus the results of my qPCR reactions.
Any thoughts on this one? Does this happen in your experiments? Thanks in advance for your help.
Best,
Carla