Nested qPCR and contamination

[Squish]

I am new to this forum, so hopefully I am posting in the correct place, and hopefully I can find a solution to my incredibly complex problem.

I have recently started a new job and have been given a nested-qPCR project. A recipe for disaster - I think so! Here is the story (sorry for it's length). I am dealing with Fowl adenovirus (DNA virus) and Avian Influenza Matrix (RNA virus) and performing a one-step reverse transcription PCR on them in parallel with a random hexamers designed for our future next generation sequencing project. The r-t reaction does not appear to be working so my boss wants to know if we are getting any r-t or if it is just input nucleic acid. So I am taking my 50 ul reverse transcription pcr reaction and splitting it into three for DNase, RNase, and no treatment. Now this is done with Avian, Fowl and negative DNA, in triplicate, with all those treatments, open tubes of products in our template addition room. Then I take these treated reverse transcription/treated products and subjecting them to our highly sensitive real time PCR assay, and I am seeing contamination up the wazoo. I am filling almost an entire plate, with all the treatments, different sample types, and testing both sets with our Avian and Fowl primer-probe combinations.

I started out with a few contaminants, then repeated it with new reagents and got more contaminants. Each time I get more contamination in my negatives, which to me indicates environmental contamination. I have to set up the qPCR in the same spot as the original reverse transcription PCR. I have to use the same pipettors. My new boss thinks I can't pipette. He thinks I am useless. The more I repeat, the worse it gets, and I am trying to convince him that using this approach is not the way to test my abilities. I have been a lab tech for 20 years, doing diagnostic qPCR assays, and am highly proven in my ability. One thing I am concerned about is that he wants me to use an automatic multichannel pipettor, and I think this is the source. It is an 8-channel pipettor, and I am only using 6 tips, leaving two channels un-tipped, which I think could be aspirating and dispensing aerosols as I move across the plate. Is this even possible?

Help! I am at a loss, and need something to work or references to convince him that this is a recipe for disaster! Any suggestions would be greatly appreciated.

[relaxin]

Are you using filtered pipet tips? If yes, I do not think contamination can come through the two untipped channels.

Do you have an AirClean workstation to set up your PCR reagents? This will help to reduce contamination.

[Squish]

Yes I am using filtered tips as well as a PCR addition hood.

The problem with using the hood is that it is in a shared area, with 5 other labs, and the traffic in the hoods is constant all day long, so the UV never gets a chance to work (not that UV is very effective anyway) at destroying DNA. In addition, protocol for the lab (it is an accredited animal disease diagnostic testing laboratory) is to run the fan in the hood for 5 minutes to blow out any nucleic acid that might be lingering and then the hood is 'safe' to use. The problem I see with the blowing part is that the open bag of tip waste is sitting in the hood, the fan is blowing stuff all over the place (ie. my PCR product that I have reintroduced) and then I go in and set up my very sensitie real time PCR.

[mchlbrmn]

Maybe you could do some wells on the side with a clean, independent, single channel pippetor.
I couldn't understand exactly what you're doing, but if you are DNAsing after RT-PCR, you can't expect it to get rid of everything. DNAse treatment of RNA is known to help, but be imperfect in removal of all contaminating mammalian genomic DNA. I don't know about virus genome?
As another thought (possibly completely off base) if could there be an issue PCRing avian genes contaminating an avian enzyme (if that's the type of RT you're using)?
If you want to see if the RT works, and can't tell because DNA virus genome can PCR, can't you tell by PCRing RNA virus or host RNA genes, with primers that can't work on host genomic DNA? The Adenovirus is spliced, isn't it? so are the primers RNA specific (versus DNA genome)? (You probably know this stuff better than me, but I'm trying to brain storm all I can).
Do you have a control of virus free host DNA?
Is this with SYBR so you can see a melt curve?