CLEMSON, South Carolina — Three Clemson University College of Science researchers recently shared their areas of expertise with a group of 16 graduate students, postdoctoral researchers and faculty members from other universities in a training workshop.

Dubbed “Camp Lasernode,” the June 12-16 workshop aimed to teach the attendees — some of whom came from outside the United States — how to replicate a lab technique called laser-capture microdissection and analyze the resulting sequence data for their own studies. The training was part of the outreach component of a $1.8 million grant researchers Julia Frugoli, Alex Feltus and Victoria Corbin received from the National Science Foundation for their study of nodule development in legumes, such as peas and beans.

Led by several Clemson faculty members, the 2017 Camp Lasernode cohort was comprised of 16 graduate students, postdoctoral researchers and faculty members from other universities.

Led by several Clemson faculty members, the 2017 Camp Lasernode cohort was comprised of 16 graduate students, postdoctoral researchers and faculty members from other universities.
Image Credit: Pete Martin / Clemson University

“Many people are on the grant that’s funding this camp. It’s a very large experiment and each member of the team directs a small part of that experiment so the team members who are actually doing laser capture in our research taught it,” said Frugoli, an Alumni Distinguished Professor and associate chair of the department of genetics and biochemistry.

“For instance, Suchitra Chavan, a postdoctoral researcher in my lab, discussed in the workshop the experiments that she does and how they work, and my lab manager, Elise Schnabel, discussed other experiments that she performs,” Frugoli said. “The instructors at the Clemson Light Imaging Facility taught about the microscope; Chad McMahan, a lab technologist, helped with making sections in the histology lab; and Dr. Feltus is doing the informatics.”

In 2016, the team began this collaboration to further investigate the symbiotic relationship between legumes and a specific species of bacteria, known as rhizobia. Along the roots of the legumes are tiny bumps called nodules that house the rhizobia, which extract nitrogen from the air and convert it to a useful form for the legumes. In exchange, the legumes provide carbon for the bacteria, which they use as an energy source to continue the nitrogen-fixing process. Since the plants are able to obtain nitrogen from the air, they can thrive in nitrogen-deficient soils without the need for fertilizer — a concept that is of interest to agriculturalists and environmentalists alike.

The grant provides funding for the researchers to identify genes in the legumes’ DNA that enable the nodules to form, a process that is unique to the plant. A greater understanding of the genes involved could pave the way for re-creating nitrogen fixation in other plants, thereby reducing the use of nitrogen fertilizers, which are too often applied in excess and pollute the environment.

The featured technique of the grant and the recent the workshop — laser-capture microdissection — uses a laser on a microscope to collect individual cells from a sample on hand (which, in Frugoli’s case, is plant tissue from the model legume Medicago truncatula). Then, genetic information contained within deoxyribonucleic acid (DNA) is gathered by analyzing the DNA product, which is ribonucleic acid (RNA).

Maria Assuncao (left) and Lucas G.S. Maia work together in the histology lab.

Maria Assuncao (left) and Lucas G.S. Maia work together in the histology lab.
Image Credit: Pete Martin / Clemson University

“Once a biologist obtains specific tissue with laser-capture microdissection, she might want to know the levels of gene expression in that sample. To find this measurement, RNA molecules can be purified from all the other stuff in the sample and sequenced using modern sequencing technologies,” said Alex Feltus, an associate professor of genetics and biochemistry whose background is in bioinformatics. “Then, supercomputers like the Clemson Palmetto Cluster are required to process the huge RNA datasets, because the number of RNA sequences that are generated these days goes into the millions. My Ph.D. student, Will Poehlman, and I taught the students how to process real RNA sequences on the Palmetto Cluster using open-source software we created that the students can use after the workshop.”

Laser-capture microdissection is a technique that typically takes weeks to set up, but for a five-day workshop, the procedures ran much like a cooking show, in order to provide hands-on demonstrations for the students.

“We condensed this long process into something of: ‘You do this, then you wait two hours, and it comes out looking like this,’” Frugoli said. “I realize, now, it’s like drinking from a fire hose. Most of these students have a specific experiment in mind that they want to do, and so they came here with the question of: ‘How would this work for me?’ We’re telling them how we do it, and we are trying to troubleshoot for them.”

At the completion of the workshop, the students left with a booklet of methods to reproduce laser-capture microdissection and five specialized microscope slides that are necessary for the technique. However, the Clemson researchers welcomed the students to return and use the Clemson microscope – outfitted  with a software interface and a laser for isolation – if they desire.

Julia Frugoli (left), an Alumni Distinguished Professor and associate chair of the department of genetics and biochemistry, gives a tour of the Life Sciences Facility.

Julia Frugoli (left), an Alumni Distinguished Professor and associate chair of the department of genetics and biochemistry, gives a tour of the Life Sciences Facility.
Image Credit: Pete Martin / Clemson University

Laser-capture microdissection is a technique that can be applied to a variety of research disciplines, such as neuroscience, oncology and plant biology. Most of the students who attended the workshop fall into the latter category, though Stephanie Dance-Barnes of Winston-Salem State University intends to use laser-capture microdissection for her studies in cancer biology.

“For my lab, in particular, we really look at differentiating between different subtypes of breast cancer,” said Dance-Barnes, co-chair of biological sciences at WSSU. “Laser capture will provide us the opportunity to look at specific tissue types or subtypes of cancer, and to be able to do more cutting-edge analyses than what we were currently doing. Right now, my laboratory is more focused on basic science, and this will be an awesome opportunity to introduce students to advanced technologies, so this workshop has been a great opportunity for me to learn some of those skills.”

Brandon Reagan of the University of Tennessee-Knoxville hopes to employ the technique in understanding how plant cells communicate with each other through channels in their cell walls called plasmodesma.

“This workshop was very informative. I’ve learned a lot about a variety of skills I wasn’t expecting to learn about, so that’s always beneficial,” said Reagan, a graduate student in biochemistry and cellular and molecular biology at UTK. “It was really helpful with learning how to process RNA-Seq data.”

Samara Oliveira is a graduate student from Brazil who is studying plant and environmental sciences at Clemson. Her biggest takeaway from the workshop was Feltus’ lessons in bioinformatics.

Elise Schnabel (left), who is the Frugoli lab manager, teaches the students about quality control for the preparation of an RNA library.

Elise Schnabel (left), who is the Frugoli lab manager, teaches the students about quality control for the preparation of an RNA library.
Image Credit: Pete Martin / Clemson University

“Right now I’m starting to work with RNA, and I had no idea about the concepts taught by this workshop. Now I know what to do with my samples,” Oliveira said.

As for Frugoli, she gives thanks to the scientists who helped her learn laser-capture microdissection, so that she can teach it to other scientists.

“In order to get the grant, I had to convince the National Science Foundation that we knew how to do it,” Frugoli said. “So, I visited other people’s labs, which can be very disruptive. Dr. John Harada at U.C. Davis was very nice and let me come and stay for three to four days in his lab, and his technician showed me everything they did, gave me all their protocols, and talked back and forth with me.

“This workshop is a way to spread what we know to every scientist so they don’t have to go through what we’ve gone through. It took us almost a year to get everything to work the way we wanted it to, and we had already done the research required. That took phone calls to people in Europe and talking to the Harada lab, and lots of back and forth like that. Now we’re trying to help other people do it, too.”

Camp Lasernode will be held once more in summer 2018. Interested applicants can visit the project’s website or contact Julia Frugoli for details.

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The researchers’ study is entitled “Spatial and Temporal Resolution of mRNA Profiles During Early Nodule Development” and is supported by the National Science Foundation under grant number 1444461. The researchers are wholly responsible for the content of this study, of which the funder had no input.