I recently ran some ddPCR using probes that have worked beautifully many times and have had two frustrating issues pop up. Firstly, running cDNA from mRNA, the positive population with my gene of interest probe is only reaching amplitudes of ~1000, despite normally reading 8000-10000. The probe is duplexed with an HPRT probe, which is reading the same as it always has.

I'm also running cDNA that's been enriched for specific miRNAs. While I would previously get a relatively clean positive signal, I'm now getting what can only be described as a monsoon, to the point that the software sometimes can't even distinguish where the positive population is. This is occurring for both my unknown and reference probes.

For clarification, the cDNA was made from RNA harvested from HEK293 cells. I have run ddPCR with these same probes on previous harvests without issue. I tried rerunning the ddPCR using previously unused aliquots of the probes, but got the same result. I have not redone the RT reaction to determine if the issue is there, but I'm hoping someone may have some insight before I give that a shot. Thanks.