Hayes describes his microarray project, and his experience visiting
the University of Wisconsin at Madison:
The focus of my summer science
project at Kenyon College
is to investigate the effect of pH on gene regulation in anaerobic E. coli cultures. In order to accomplish this, I used microarray
technology, which allowed me to investigate the entire E. coli
genome. A related project the previous year, conducted by Lisa
Maurer '04, was just accepted for publication in Journal of Bacteriology.
My aim was to extend Maurer's approach to study pH regulation under
anaerobiosis. We ultimately found some remarkable differences, particularly
in the regulation of catabolic enzymes and flagellar biosynthesis.
At Kenyon, I worked with
professor Joan Slonczewski and research fellow Liz Yohannes to grow
anaerobic cultures of E. coli, from which I extracted, isolated,
and cleaned the RNA. To run microarray
experiments, I went to the Gene Expression
Center at the University
of Wisconsin-Madison. During
my one-week stay in Madison,
I was able to complete the aspects of my experiment that could not be
done at Kenyon. I also had the
opportunity to work closely with Sandra Splinter, whom I learned a great
deal from as I followed her AFX training program.
When I arrived
at the airport in Madison
on Saturday, I was met by Brad Hersh, a Kenyon alumnus who did his PhD
with Bob Horwitz at MIT. Brad
is now a postdoc at University of Wisconsin-Madison.
After checking into the Frederick
Center, he gave me a tour
of the city. We spent a good
part of the afternoon walking around Madison
and I got to see a number of places, including the student union, the
capitol, and the art fair that was going on that weekend. On Sunday I walked to one of the many parks
in Madison where there was
pick-up Ultimate Frisbee. It
was a fun group of people playing and I met some interesting people.
Brad also
brought me to his lab so that I could see the research he was working
on. He is currently doing a study on the evolution
of the spots on the wings of butterflies. It was interesting to see his lab because it
differed so much from the labs at Kenyon.
The first difference that I noticed was that here at Kenyon,
our labs have a lot of open space. Everything
there was crowded in, and there was little room to spread out your work. Another difference between this lab and those
at Kenyon was that there more high-tech equipment. This is also something I noticed once I saw
the equipment at the Gene
Expression Center.
At 8:00 AM on Monday morning, I arrived at the Gene
Expression Center. I met Sandra and Wayne, the people helping me
with my research. I had a quick
introduction to the center and the work that they do and Sandra and
I discussed my project. That
day we began synthesizing the cDNAs from my RNA samples.
Sandra was very helpful teaching me about how and why the reactions
occur and Wayne taught me
about the chip hybridization and scanning.
It was exiting to see how the gene chips are run and analyzed. I also got to see some spotted arrays that other
people were running in the lab.
Of particular interest was the equipment that they had in the lab.
I was especially fascinated with the NanoDrop Spectrophotometer,
which allowed you to read the absorbance of your sample by only taking
a microliter of sample and dropping it onto the reader.
This was much faster and easier than using traditional cuvette
readers that require you to dilute your sample.
The fluidics station, which automated all of the staining and
rinsing steps for the microarray chip, was also intriguing.
It allowed for multiple arrays to be run at once and it automated
all of the steps so that they are all precise and easy to run.
There were many pieces of equipment there that I had never seen
before and it was very exciting to be able to see and use them.
The samples that I isolated at Kenyon had been sent to Madison
so that they were there for me when I arrived.
Sandra and I worked together on the AFX protocol while I was
there. The first step in the protocol is to synthesize
cDNA from the RNA samples. This
is done by priming the DNA with random hexamers, followed by reacting the mixture with a cocktail that contains dNTPs that
extend the cDNA from the primers, completing the cDNA strand. We used a thermocycler to incubate the reactions
to ensure consistent heating.
After the cDNA reaction is complete, the RNA is digested and the cDNA
is purified using a QIAQuick PCR purification kit.
In order to verify that enough cDNA was made to continue the
reaction, the concentration is determined using a NanoDrop Spectrophotometer.
Next, the cDNA is fragmented to get an average length of 50-100bp. The incubation time for the fragmentation reaction
varied for each sample depending on the concentration. From earlier work, Sandra was able to approximate
the fragmentation time needed for the different samples. The fragmentation lengths are checked on an
agarose gel to ensure that the cDNA fragmented to the correct length. The samples that are fragmented correctly are
end-terminusly labeled with biotin.
At this stage, the samples are ready to hybridize to the microarray
chip. All of the steps are automated to ensure consistency and accuracy
across all the arrays.
The last step is to scan the gene chip and extract the data.
These data were sent to Kenyon for further analysis.
At Kenyon, I am processing the data with the help of Michael
Radmacher, statistician at Ohio
State University. With his help, I am able to perform complex
analyses in order to interpret my data and to classify groups of expressed
genes.
At each
stage of the experiment at Madison,
some of the samples needed to be re-run.
Either there wasn’t enough cDNA made or the cDNA was over or
under fragmented. In these cases,
we had to go back and synthesize new cDNA for that sample and start
over. This taught me that in experiments such as this
one, you can’t expect everything to work correctly the first time. Even though we tried to minimize the variability
between samples by making cocktails of reagents and by being as careful
as possible, a few samples from each set didn’t work. For the microarrays, 14 out of 15 were successful,
so the last one had to be redone. In
order to isolate the problems, Sandra taught me to keep track of the
lot numbers of all of the reagents and supplies so that you can identify
trends in the failed reactions and possibly link
them to bad reagents.
Outside of the lab, at Madison, I spent a lot of time
walking around, exploring different areas of the campus and the city. I also got a chance to try some of the local
ice cream that is made at the campus dairy farm. Before visiting, I knew very little about the
school, but now that I’ve been there, I think that it would be a great
place to go to school. I am now
thinking about schools that I want to apply to for medical school, and
I think that the University of Wisconsin-Madison would be a good place
to apply to. This trip has allowed me to experience aspects
of research that we do not have here at Kenyon, and it gave me the chance
to visit a great school that I am now thinking of applying to in the
future.
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