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.