Hi everyone,

If somebody can explain to me what's happening to my samples it would be great !

I'm currently testing a RT-qPCR for three primers, one of wich can amplificate genomic DNA if present. (not a multiplex)

On the first day, i do a RNA extraction, I degrade genomic DNA by ezDNase, and then i made a RT with a NRT. To confirme no genomic DNA contamination, I do a qPCR with only the NRT. No amplification.

The second day, two qPCR :

First with different concentration of primers. Here no amplification in NRT and amplification in RT. Good

Second this time with different temperature. Here some amplification in NRT but not high enough to be a problem (under 100 RFU) and amplification in RT. Still Good

The third day, one qPCR :

Different concentration of RT and NRT for standard curves. And then big amplification in NRT for all well.

That amplification, is only on this specific primer so no contamination of the NRT with RT.

NTC without amplification, so no contamination on the mix.

the Cq NRT < Cq RT so no mistake of sample (the Cq of RT are then greater than the previous qPCR)

I suppose that I either cross-contaminated wells or had DNA first and then contaminated my NRT with some mix previously.

I start a new extraction and do the same as the first day, no amplification observed in all NRT but one. I'm careful to not contaminate my sample with mix.

I wait for the week-end and do qPCR with my samples : NRT amplificate in every sample ! still good NTC, no amplification for the NRT of others primers and most of all, I have a NRT whose Cq is bigger than the RT so no cross contamination.

I suppose that I shouldn't do the first qPCR for verification sake only, as it will not change at the same rate as the RT.

But, can somebody give me a logical explanation for why my NRT conserved at -40°C seems to amplificate with time ?