Recently we've been having trouble with cDNA contamination in -RT controls for both qRT-PCR and RT-PCR. Our primers are designed around a large intron, so the gDNA product is much larger (about 800bp) than the cDNA product (about 180bp). When we run PCR products of the -RT control on agarose gels we get a product the size of the cDNA product. Our No-template control (primers and PCR reagent only) is always clear, so our PCR reagents are not contaminated. And we are definitely not putting RT into our no -RT controls.
Today I took some old cDNA samples out of the freezer and ran a RT-PCR. Previously when I ran these samples on a gel the -RT was clear. Today it was contaminated (but NTC clear). All I did was take the cDNA out of the freezer and pipette it into the reaction.
Are there any suggestions as to where my cDNA contamination is coming from?
Any help will be appreciated, we are stumped!