Hi. I'm doing qPCR on healthy viroid sample. I got N/A or zero value during my amplification but some of my friends got cq value for healthy samples. It make me worried. Healthy sample suppose have cq value or not? Thanks.
Cq is the quantitation cycle which is the first cycle whereby detection of the fluorophore released during amplification is detected. If you are running a lab experiment and the DNA that is specifically targeted by your probe was in the mix, it should have a Cq.
Many things could have gone wrong as PCR can be quite sensitive and beginners tend to do a few tries before they get it right. Did you run the same number of cycles as everyone else? Did you have the same quantity of DNA in the mix? It could be poor pipetting or you forgot to add any component to the mix (polymerase, nucleotide, etc).
Sorry I just realised I answered in a rush and it may not have been clear enough. I did not quite understand the first part, is your sample healthy? If you are using the qPCR to detect viroid in healthy samples then it would not be present.
The important data here is not "Cq/no Cq", it's what that Cq value IS.
PCR is a very, very sensitive technique, and uses exponential amplification: any trace mispriming/primer dimer events only really need to occur ONCE to produce a viable (but incorrect) template for subsequent exponential amplification. Essentially, if you run a PCR for long enough, you'll almost always see SOMETHING.
The beauty of qPCR is it tells you WHEN that something appeared, and thus how much template you started with. If you are performing PCR for viral DNA in samples that should have no virus, then you should (ideally) have no amplicon at all. You may however still see signal from primer dimer, mispriming, or trace contamination. And this may be 'freak event' based rather than predictable.
For example, you may see no Cq value (no amplification), but someone else doing the exact same PCR might see Cq values of 35, 36 and 'no amplification': all of these are statistical noise/trace contamination. 35 cycles is enough to amplify even a single template molecule to saturation, so the conclusion is either "there was ONE viral genome present", or (more realistically) "it is noise".
Conversely, if you do the same PCR for infected tissue, you may get Cq values of 25 or so: a thousand times higher, effectively. This is NOT noise.
So: yes, you can get amplification even from negative samples, but you can use the Cq value (and melt curves/gel electrophoresis) to distinguish noise from signal.
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