We have a Q5 system and use a one-step qRT-PCR using software designed primers and probe (contains FAMA reporter, Zen quencher, and Iowa Black quenchers). Using either Luna one step probe qRT-PCR reagents or Extaq one step qRT-PCR reagents, we get decent signals on our targets (20-34 CT) and our water plus pcr reagents only negative controls come up as undetermined. However, when we use irrelevant RNA, we get a signal around 30-35. It does not matter what irrelevant RNA we use and we get the same issue if we just use DNA in our PCR without the RT step using irrelevant DNA. Given the controls are loaded first and away from our samples and the water only gives no signal, we cannot understand why we get false amplification using irrelevant RNA or DNA. The curves for the water only are a flat line while the irrelevants look like they typical S curves that sample targets give. This has happened in a number of different primers and different RNA targets for us. Conditions are run with what either PCR reagent company says using 95 for 10 sec and 60 for 30 sec with read and a RT prior step at 50 or 55 for 10 minutes and then 1 minute at 95 before the actual detection cycles begin. The targets are viral genes and we use a different virus which doesn’t have the genes of interest as the irrelevant rna.