Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?
This is done because at some point you start amplifying nonsense and not get a meaningful signal. If (and this is most often not the case) you have the ideal doubling of you DNA template in each cycle, you can calculate how little DNA you start with to detect it only around cycle 40 - or the other way round: What you will amplify and see as a &quot;signal&quot; by then.
If you have templates which always really come this late, I would use more DNA (and probably more RNA to start the rtPCR) until this comes at least 2-3 cycles earlier and then do serial dilutions of your template to see if this signal is real or not. If so, you should see a relation between Ct and dilution.
For doing this you need a negative control which stays really negative until your sample signal shows up - which is tricky on its own because at some point you will see some kind of primer self-amplification. If this occurs in the same range as your sample, your signal cannot be used. If no other primers can be used for technical reasons, sometimes nested PCR has to be considered.
And last but not least all your reagents degrade over time by cycling them 40 times through the PCR program (also the polymerase), which affects the efficiency of the reaction.