Trouble with microarray. Too much variability between biological repeats - (Sep/14/2012 )

Alright I'm going into my 2nd year of grad school and for one of my experiments I'm doing a microarray on swarming cells in P. aeruginosa. Swarming is a type of motility in P. aeruginosa, and I'm doing a microarray of the wild type vs. a mutant which hyper swarms. I've had to work out some problems but I finally got the phenotypes I wanted. Basically I do everything up to and including the genomic DNA check after RNA extraction and DNase treatment, at which point a technician takes over and does the microarray.

I've attempted this twice, but each time there was too much variability between each of my biological repeats (P-values were too high) and this has eaten up ~8 months of my time. Are these types of problems normal? I feel like such a straggler right now but I'm still trying. I'm on my 3rd attempt, and I really have no idea what's going on. Here's a basic outline of what I do,
- Inoculate my swarm plates and let my mutants swarm for 18 hours
- Collect the swarm cells with a swab and "add" the cells to water + buffer by swishing the swab in the solution
- Add 2 volumes of RNA protect
- Centrifuge for appropriate amount of time
- RNA extract using QIAGEN RNA extraction kit
- DNase treat with Ambion DNase1
- Genomic check w/ rpsL

I've made sure all the conditions were the same, each BR was treated exactly the same to the best of my ability. I really don't know what's going on, so any help/suggestions would be helpful.

Thank you

You did not mention technical repeats. Have you included them, if yes, what is the variability between them? Without first knowing variability between technical repeats, it is hard to see what caused the variation between biological repeats.

pcrman on Fri Sep 14 15:54:12 2012 said:

Technical repeats weren't included, if there was variability between technical repeats, then I assume the problem is coming from the technicians end. But she has done microarrays on swarm cells before that have worked, so I think the problem is coming on my end. I just have no idea what is causing it since my part is so straight forward.

I've been thinking about this and the only thing I can possibly think of is that during the DNase treatment, I add DNase inactivation reagent, mix it, then centrifuge. I'm using the kit from Ambion. After centrifugation, the inactivation reagent forms a white "pellet" at the bottom. But I've noticed that it's quite "loose" and that it is disturbed quite easily. I try to avoid carryover, but there isn't any guarantee as it is quite difficult to tell.

I've been told you don't want carry over as this could inhibit other enzymes etc. If some of my biological repeats have this DNase inactivation reagent as contamination, I'm guessing this could lead to variability with my biological repeats?

Any other suggestions?