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Alison Elizabeth Murray Sees Microarrays at Extremes of the Globe

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At A Glance

  • Alison Elizabeth Murray
  • Assistant Research Professor, Division of Earth and Ecosystem Science, Desert Research Institute, Reno, Nev.
  • 1989 — BA, Biochemistry, California Polytechnic State University.
  • 1995 — MS, Molecular and Cellular Biology, San Francisco State University.
  • 1998 — PhD, Ecology, Evolution and Marine Biology, University of California, Santa Barbara.
  • Postdoc — Microbial Functional Genomics, Michigan State University.
  • Research Interest — Developing environmental genomics technologies for studying gene expression in organisms sampled directly from the environment. Current projects are: gene expression in Antarctic bacterioplankton in constantly cold waters; gene expression of episymbiotic microorganisms associated with the hydrothermal vent worm, Alvinella pompejana, and developing phylogenetic arrays to detect microbial diversity and activity in carbonate hot springs.

When you have visited the Antarctic enough times, you often get to have your own room. Having been there seven times, Alison Elizabeth Murray has earned that distinction. Good thing. Next time, she wants to take another piece of luggage with her, her 30-pound microarray scanner.

Murray is among a group of pioneers working to take microarray-based analysis out of the lab and into the field: The 36-year-old molecular microbial biologist, a native of Carmel, Calif., has also brought microarray research techniques to the Desert Research Institute in Reno, Nev. In her new lab, Murray prepares microarrays to study gene expression in organisms sampled directly from the Antarctic Sea, or those collected from the Alvin submersible diving to the hydrothermal vents in the Pacific Ocean, and soon, those in the high-temperature carbonate springs around Yellowstone National Park and near Siena, Italy.

BioArray News recently caught up with Murray to talk about her research and microarrays.

I understand we are catching you just getting back into the lab after an Antarctic visit.

This is a good time for me to come home. We have our first sets of environmental arrays and we have all these freshly collected samples, everything has come together. We have positive results from doing DNA hybridization experiments with our environmental DNA samples. We can detect Archaea, my big group of interest. They are planktonic, cold-loving microbes and are really prevalent in the water column in the Antarctic winter-spring surface waters.

The first microarray we are working with has about 80 archaeal genes on it. So, we know we can detect, at least, the DNA to those genes hybridizing to the Antarctic samples that we collected last year. Within the next couple of weeks we are going to be doing the RNA experiments.

All of our sequences come from an environmental library of plankton collected in Antarctica and developed a couple of years ago. We are sequencing seven fosmids from different bacteria to get the environmental data sequence to put on the microarray. We have only a couple done so far. We can then query the arrays for both gene presence and expression with differing environmental samples.

I’ve been sampling on different kinds of environmental gradients, for example, different levels in the water column, or different seasons. This year we were [in Antarctica] between the change from winter water to spring water conditions. The carbon concentrations in the water column change dramatically during this time, and the bacteria respond significantly in their activity levels — as they finally have something to grow on as a result of the spring phytoplankton blooms.

We are curious to know: Does gene expression change if you all of a sudden add 10 micromolar carbon to the water column? So, instead of doing the experiment in the lab, we can let the environment do the experiment. That’s the big question out there: If we can learn something about how these organisms respond to environmental change and whether we can use microarrays as a tool for that. I don’t think we know yet. We are right on the cusp of being able to figure this out.

How did you get involved in microarray research?

In grad school, I did a lot of membrane hybridizations. We were always limited by the space that we had on our membranes — the controls took up more space than the samples we could put on them. When I started reading about microarrays, I thought, ‘Oh wow, we could put a lot more information down, instead of having the whole thing dominated by the controls.’ [So] I was interested in doing a postdoc and learning about microarrays. I found a good one at Michigan State University at the Center for Microbial Ecology. I worked with Jim Tiedje, the director. He had the same vision of wanting to get involved in microarrays and could see that there could be a number of avenues where microarrays might be useful for microbial ecology studies.

So, you are a self-spotter?

Yes, I don’t work on any model organisms at this point, so that is the only route to go.

What is your laboratory like?

I call it a molecular microbial ecology lab. We have all the equipment here to prepare the materials for spotting microarrays and do the hybridizations, all of the hands-on specifics for doing the experiments. And, we just got an award from NSF-EPSCoR providing funds for a microarray scanner, so I acquired an Axon GenePix 4000B. The UNR campus has a Virtek spotter and a technician who runs it.

What are the challenges to doing microarray analysis in the field?

I have been going down to Antarctica for the last two years to Palmer Station, collecting [water] samples, then concentrating [them] and doing some RNA extractions. I would hope that if we get [our grant] renewed to go back down to Antarctica, I could take my scanner with me.

The Axon scanners are a manageable size, though they aren’t exactly portable. Still, I think I can bring [mine] with me or ship it down. For the hydrothermal vent research, our goal is to try and do the actual hybridization experiments out at sea. We think that the advantage is to learn while we are sampling, and be able to adapt our collection strategies, so we can go back down to the vent and collect more at one area, or more of one kind of worm. If we are doing it well at the lab, then, we can feel good about going out to sea. Still, it is a challenge to [be] doing this on a rocking ship.

So, collection is the gating factor because you can’t culture the organisms you study.

In both cases, at the hydrothermal vents, or Antarc-tica, you can only go once a year, if you are lucky. For the vents, you’re fortunate to get three weeks on a ship. So the time is so precious for collection. With the logistics of getting to Antarctica, it makes more sense to stay there a little longer: I was there eight weeks this year.

How do you go about data analysis?

In the past, I have [used] the Michael Eisen software packages. I got hold of the GenePix software and have been really impressed, I use it for image analysis and will continue to use it. When we got the scanner, we also obtained the new Acuity package. I usually normalize the data by doing it in a spreadsheet. But, I’m planning on trying to do it all on Acuity if possible. GeneSpring is on the back burner, if we can’t do it in Acuity. The costs are pretty high though. Being in a group of four people, paying $5,000 or more for software is hard to justify. I’m into finding programs that are freely available and actually work. We are trying some of those.

One of the challenges that I enjoy, with working in this interdisciplinary area between being a microbial ecologist and trying to use genomics technologies, is figuring out how to work up the data. My questions are different than if I had the E. coli genome and I was trying to compare growth on two different carbon substrates. Our arrays are going to be very different. In some cases, we perform more than one experiment at a time on a slide. If you analyze all the data one way, the norm for most programs, you assume that all ratios should be 1 and you normalize data to that. For some of the experiments that we are doing, we have no idea that the ratios should be 1. So we do more spiking controls, rather than just normalizing the data to itself.

What improvements do you want for microarrays?

Labeling technology has to improve. Highly sensitive labeling technologies are needed for microarrays to work in the environment. We can’t always get 10 micrograms of RNA from a sample. That’s a lot if you are collecting from the environment.

We are collaborating with Genisphere: They have one flavor of labeling technology that I am hoping will increase the sensitivity of detection. In that case, they have a molecule (dendramer) that binds lots of dye. You can have 10-fold greater signal than if you just did a regular reverse transcriptase-dye incorporation reaction. Any way to more sensitively detect samples being hybridized, I think, is going to be the make-or-break point about how useful arrays will be for a majority of environments. We are lucky because working in the oceans, you can get a lot of sample by filtering and filtering water. We collect 200 liters of water and concentrate that down to about 500 milliliters, then we basically get a bacterial soup. From that, we might get 40-80 micrograms of RNA, and that’s a good sample. For other samples, the hydrothermal vents, getting enough sample is going to be a challenge.

 

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