Between 30 and 90% of the dust in many rooms is sloughed human skin cell material... and in my building, that dust is also intermingled with cow, horse, mouse, rat, rabbit, pig and sheep dust... Any reference gene I've tried to amplify whose sequence is identical or highly similar among any of the animals mentioned above, consistently give a background reading in the NTC well (anywhere from 36 to 38 cycles). If your problem is not "dust" - you may be dealing with primer dimer in your NTC (if using SYBR Green-based qPCR). These dimers do not form as much in your other reactions in which actual template exists. If you are using a probe-based (hydrolysis probe) assay, your NTC signal is most likely room contamination. "Your NTC for cDNA" statement seems a little curious though - since an "NTC rxn" often implies that water (and not an un-related cDNA or any cDNA at all since it is a "No Template Control") is being used as sample in those reactions. What does your NTC reaction consist of? Also, if your contaminating signal is at least 6 or more Cq away from the 'legitimate' signal in your samples, the additive quantitative effect is negligible.

Thank you for your reply. The cDNA NTC consists of revers transcriptase, water and master mix buffer with random primers. My assays are probe based. For a couple of assays the Cq is less than 6 Cq's away from my 'legitimate' signal of my samples. If it were dust, I would expect my other NTC which consists of water, mastermix and assay also to be positive?

It appears, then, that some sequences among the random primers anneal to the 5' end of your probe and summon low-level amplification and cleavage by Taq 5' exonuclease activity.. Are your random primers hexamers, ocatmers, nonamers? If this is a One-step process (wherein the mastermix contains both RT and Taq enzymes) I would think that just dNTPs would be used (as opposed to random primers) in the mix. Given the large number of possible sequences present among your random primers, you may be inadvertently complementing the sequence of your probe and causing a reaction to occur. Perhaps this particular breed of 'NTC' doesn't serve its intended purpose here. I would just look at your NTC reactions which use water as "sample," and, if those are clean, your good to go.