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There are 6 pages to this Feature:
Molecular Approaches to Neuroscience - The Main Course - A Satisfied Customer - Q&A - BTC - Photos & References
"...I had a much better understanding of the language of the techniques so that I could better interact with the people in the lab who were doing the work."
--Allan I. Basbaum, UCSF
Many scientists liken working in a molecular lab to cooking. The conventional wisdom is that the gourmet, perhaps even the gourmand, makes for a better bench scientist. The reasoning? Well, much molecular lab work resembles cooking---from preparing the reagents, cultivating cultures, measuring mixes and reactions, knowing how and how long to heat the "dish." The metaphors rise like well-leavened bread. Although not always pleasing to the sense of smell, the molecular lab is definitely recognizable by its redolent odors of bugs, buffers and chemicals.
Lih-Fen Lue carefully loads sample into a protein minigel. Attempting to correctly deliver a few microliters of a precious sample into a nearly translucent well only a bit wider than a pipet tip is a challenge at first try. After the umpteenth gel, though, sample loading becomes second nature. The tools of molecular biology involve exact sample manipulations, which can bring any scientist to his or her knees, literally. It's just one right of passage into the esoteric world of molecular biology.
If you're not a natural cook, the molecular lab can be a daunting place---populated by so many unique utensils and fresh ingredients ready to spoil and not far removed from the watchful eyes of cantankerous diners (a.k.a. granting agencies) who will only pay for the best cuisine. The BioPharmaceutical Technology Center Institute (BTCI) and Promega Neurosciences offer a training course in recombinant DNA technologies for classically trained neuroscientists wanting to learn molecular biology.
Early this summer, science students from across the United States congregated in Madison for five days of intensive training and instruction in Molecular Approaches to Neuroscience. From the classically trained neuroscientist to the non-neuroscientist, these students gathered to hone their laboratory skills in molecular biology and to learn new ones.
Most came to learn, some to improve, but all wanted to enhance their practical
understanding and hands-on use of molecular techniques. Mary Behan, a neuroanatomist at
the University of Wisconsin, 
enrolled to "learn the language of molecular biology...the readings,
lecture presentations and finally seeing what it was all about. It took the mystery out of
molecular biology." This sentiment was echoed by many of the class participants.
Mary Behan, University of Wisconsin, preparing samples for electrophoresis.
Mary is currently studying how age and gender affect the brain serotonergic system, especially the areas involved in sleep and breathing disorders, and is interested in furthering her training because she "doesn't want to be technically limited." Learning these molecular techniques first-hand now allows her to better interact with colleagues, whether on a daily basis at her institution or at national meetings such as the Society for Neuroscience Annual meeting. An additional bonus of the course has been the initiation of collaborative studies with fellow neurobiologists. There's no doubt that new techniques enhances networking opportunities.
"Participating in the course took the mystery out of molecular biology...I lost a lot of my intimidation."
--Mary Behan, University of Wisconsin
Who Came to Dinner?
The advanced scientific course was held at the BTC (on the campus of Promega Corporation) in Madison, Wisconsin. Although each student presented a unique background and training in science, all came together to learn. Students from industry and academia came to enhance their knowledge and understanding of, not to mention practical experience using, molecular techniques.
The 1999 Molecular Approaches to Neuroscience class: (back row) Dr. Mary Haak-Frendscho (instructor), Brian Pike, Ed Plowey, John Woollard, Chris Ahern; (third row) Joe Beatty, Anne Ratashak, Valerie Schutzkus, Gaylen Edwards, Jeff Kramer; (kneeling, second row) Changying Ling, Lynda Wright, Kristi Buxton, Steve Fredman, Fernando Cardozo-Pelaez, Rick Salatino (instructor); (kneeling, front row) Laura Scott, Mary Behan, Lih-Fen Lue, Amy Prevost (instructor).
One popular menu item: reverse transcriptase-PCR, or "RT-PCR." Students isolated RNA from mouse and rat brain tissue using the SV Total RNA Isolation System(a), and amplified specific messages for NGF and beta-Actin, not an atypical technique in many neuroscience laboratories today. See the eNotes applications article, "Measuring Gene Expression in Mammalian Brain Tissue" for more information.
The instructors lectured on the salient points of the topic of the day, and then the class proceeded to the benches in the teaching laboratories to actually perform and troubleshoot the procedures for themselves.
From Hot-Plate to Industrial Range Cooking
Can a five-day course turn a neuroscientist into a "card-carrying" molecular neuroscientist? Well, no. Or, "NO!," as some students made perfectly clear (see Q&A).
But the seeds of understanding certainly can be nurtured. Students picked up many of the skills needed to design and perform experiments using molecular techniques---handling RNA, accurately constructing a reaction mix and loading samples in delicate gels perched in awkward electrophoresis equipment: Tools that allow the neuroscientists to open up many new possibilities for dissecting their specific scientific questions.
See the section, Q&A, to see what the students thought of the tightly integrated lecture/laboratory exercises.
This story continues with "The Main Course."
There are 6 pages to this Feature:
Molecular Approaches to Neuroscience
- The Main Course - A Satisfied
Customer - Q&A - BTC - Photos & References
The hands-on laboratory and lecture course, Molecular Approaches to Neuroscience, offers classically trained neuroscientists experience measuring gene expression in relevant tissues. Promega scientists cover many molecular techniques with applications in neuroscience research. Specific topics include: RNA purification, RNase protection assays, PCR(b), RT-PCR and quantitative RT-PCR, T-vector cloning, Northern blotting, transcription reactions, and Western blotting and ELISAs.
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The next Molecular Approaches to Neuroscience course is June 11-15, 2001. Contact Rick Salatino (608-274-4300; FAX: 608-277-2516) for additional course content information, or register online. |
Rapid PCR product ligation using the pGEM®-T Easy Vector System and the new 2X Rapid Ligation Buffer.
There are 6 pages to this Feature:
Molecular Approaches to Neuroscience
- The Main Course - A Satisfied
Customer - Q&A - BTC - Photos & References
Allan Basbaum was a student at the first offering of Molecular Approaches to Neuroscience in the summer of 1995. Allan enrolled in the course, as he puts it, "to find out whether it [was] a direction that I wanted to take." Upon completion of the molecular techniques training, Allan returned to his laboratory at the University of California-San Francisco and began implementing the techniques in his neuroscience studies. A few years (and experiments) later, Allan and his colleagues produced a PPT-A knockout mouse and in 1998 reported the findings on the involvement of PPT-A and substance P in pain reception in Nature.
"It's not that I learned how to make the mice, but that I had a much better understanding of the language of the techniques so that I could better interact with the people in the lab who were doing the work."--A. Basbaum, UCSF
Briefly, substance P was suspected in the signaling of pain, but its precise contribution and how it interacted with other putative "pain" transmitters, notably glutamate, was unclear. Substance P is encoded by the gene, preprotachykinin A (PPT-A), as is the related tachykinin, neurokinin A. In their knockout mouse, pain in the moderate to intense range was abated significantly, as was the neurogenic inflammation associated with the peripheral release of substance P and neurokinin A. Their conclusion: the release of tachykinins from primary afferent pain-sensing receptors, or nociceptors, is required for pain emission in the moderate to intense range.
[Click on figure to view higher resolution image]
'Pain' responses to different thermal or mechanical stimulus intensities.
Panel a, Licking/jump latency in the hot-plate assay (n=12); at 55.5°C the homozygous mutant mice (black bars) showed a decreased pain response compared to wildtype (white bars). Panel b, Paw withdrawal latencies to noxious thermal stimuli (n=8): at midrange intensities (8.0 volts), mutant mice also showed a decreased pain response. Panel c, Withdrawal threshold to a mechanical stimulus (von Frey hair; n=16); wildtype and mutant mice do not differ. Panel d, Response latency to tail clip (wildtype: n=31; homozygous mutant: n=21); the pain response of the mutant mice is significantly delayed (**, P < 0.01) (1).
Figure reprinted from Nature (1998) 392, 390, by permission of Macmillan Magazines, Ltd., and Dr. A. Basbaum.
There are 6 pages to this Feature:
Molecular Approaches to Neuroscience
- The Main Course - A Satisfied Customer - Q&A - BTC - Photos & References
The 1999 Molecular Approaches to Neuroscience class.
"I now see how I can use molecular techniques to get answers to questions relevant to my research. I am also better able to evaluate research that uses these techniques."--S. Fredman
"Things are much more do-able and within my technical reach."--C. Ahern
"A better appreciation for what is involved in each step of the assay. I've had techs running these protocols and really had to rely on their experience or someone else to help with troubleshooting. I understand the technical aspects better now."--G. Edwards
"There is nothing unique about applying molecular biology techniques to neuroscience. However, nervous system tissue may be more demanding than others. For example, particular messages (mRNAs) may only be expressed at very low levels. This is a traditional signal-to-noise problem, but with a new twist."--S. Fredman
"From my standpoint, the unique aspect of applying molecular techniques to neuroscience lies in the multi-level approach we can take in the lab. We can apply whole animal, in vitro electrophysiological and molecular techniques to provide a genomic and environmental explanation for behavioral, and in my case autonomic, phenomenon."--J. Kramer
"In my case it is the lack of homogeneity across cells in the brain. Trying to sort out which cells are involved in specific actions is a big task."--G. Edwards
"I think the lecture-lab approach is necessary...becasue there is so much theory behind many of the techniques. It is vital for a molecular biologist to first understand why something need be done and approach the details on a case-by-case basis."--K. Buxton
"The tight lecture-lab approach [was] extremely effective."--S. Fredman
"I liked it because it offered the opportunity to ask questions about each step of the process. If you didn't understand why you did something, the lecture let you ask about it."--G. Edwards
Gaylen Edwards, University of Georgia, prepares a protein minigel for electrophoresis and subsequent Western blotting."I believe that the approach is a good one in that the theory of the technique was taught and then we actually went and performed the technique right away. Because of that, I feel there is an advantage over a course done at the university where things get stretched out over the semester and lecture and lab are not necessarily very close together..."--J. Woollard
"NO! But coupled with curiosity and a willingness to keep learning, it is a great springboard to becoming a molecular biologist."--G. Edwards
"NO! However, I think that an appreciation for the lab work is developed and the seeds for ideas and the potential for new approaches to many problems are made plain."--J. Woollard
"No. It is such a vast field. An intensive training course would be a nice introduction to the molecular world, but there is too much information to approach/absorb in a short period of time."--K. Buxton
"No, one does not become a molecular biologist (or any other kind of researcher) after only one week. What this course did was expand horizons. It made molecular biology much less intimidating..."--S. Fredman
"Using the supplied handbook and consulting people on campus. The course work won't really solve my problems, but now I know what questions to ask."--C. Ahern
"I will look back on my notes and consult colleagues who perform these techniques on a regular basis. Also, I will check online resources as well as published notes."--J. Kramer
"I can be a more active participant in the troubleshooting."--G. Edwards
[Editor's note: see Photos & References for these recommended reference materials.]
"Maniatis and Sambrook is a classic."--C. Ahern
"I will start with the books noted in class and other molecular techniques books located in the library and other labs in the department."--J. Kramer
"We use anything that we can (Maniatis, Promega Notes, Clontech notes, etc.). You have any good suggestions?"--G. Edwards
"There are several protocol books available from the library and in our lab such as Current Protocols, and the book list given to us in the course, as well as several other books..."--J. Woollard
"Several reference materials were recommended in the course. There are others that have also been recommended. My colleagues have some of these (i.e., the Protocols on CD-ROM)."--S. Fredman
There are 6 pages to this Feature:
Molecular Approaches to Neuroscience
- The Main Course - A Satisfied Customer - Q&A - BTC - Photos &
References
The BTC, or BioPharmaceutical Technology Center, is home to Promega Corporation, Ophidian Pharmaceuticals, Inc., and the BioPharmaceutical Technology Center Institute (BTCI). BTCI offers basic and advanced courses in scientific education for the novice student to the professional scientist. Programs are developed and implemented in collaboration with Promega and other corporate sponsors, as well as academic institutions. Teachers and instructors from industry, Promega and educational centers near and far collaborate on the various offerings, affording students qualified and experienced educators in specific disciplines.
For a listing of upcoming advanced courses and additional information, please visit the BTCI home page at: www.btci.org/.
There are 6 pages to this Feature:
Molecular Approaches to Neuroscience
- The Main Course - A Satisfied Customer - Q&A - BTC - Photos
& References
Lih-Fen Lue, Sun Health Research Institute, loading an electrophoresis gel.
Terri Sundquist, course instructor and Promega Technical Services Scientist, proudly displays the results of the Northern blots (of 18s RNA and G3PDH) to the class. We got signal!
Fernando Cardozo-Pelaez, University of South Florida (left) and Brian Pike, University of Texas-Houston (right).
Molecular Biology
Protocols and Applications Guide, Third Edition, Promega Corporation.
Current Protocols in Molecular Biology, Ausubel, F.M. et al., eds.,Wiley & Sons, NY.
Molecular Cloning: A Laboratory Manual, (1989) Second Edition, Sambrook, J., Fritsch, E.F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
LabFax: Molecular Biology Second Edition Vol. I, Recombinant DNA, (1998) Brown, T.A., ed., Academic Press, NY.
LabFax: Molecular Biology Second Edition Vol. II, Gene Anaylsis, (1998) Brown, T.A., ed., Academic Press, NY.
Neuroscience
Current Protocols in Neuroscience, (1997) Crawley, J. et al., eds., John Wiley & Sons, Ltd.
Neuroscience LabFax, (1997) Lynch, M.A. and O'Mara, S.M., eds., Academic Press, NY.
Experimental Neuroanatomy: A Practical Approach, (1992), Bolam, J.P., Oxford University Press, Inc.
Neural Cell Culture: A Practical Approach, (1996) Cohen, J. and Wilkin, G.P., Oxford University Press, Inc.
Immunology
Current Protocols in Immunology, (1992) Coligan, J.E. et al., eds.,Wiley & Sons, NY.
Molecular Immunology, Second Edition, (1996) Hames, B.D. and Glover, D.M., eds., Oxford University Press, Inc.
Amplification
PCR1: A Practical Approach, (1991; reprinted 1994) McPherson, M.J., Taylor, G.R. and Quirke, P., eds., Oxford University Press, Inc.
PCR2: A Practical Approach, (1995) McPherson, M.J., Hames, B.D. and Taylor, G.R., eds., Oxford University Press, Inc.
PCR3: A Practical Approach, (1998) Herrington, C.S. and O'Leary, J.J., eds., Oxford University Press, Inc.
PCR (Introduction to Biotechniques), Second Edition, (1997) Newton, C.R. and Graham, A., eds., Spriner Verlag.
PCR Cloning Protocols: from Molecular Cloning to Genetic Engineering, (1996) White, B.A., ed., Humana Press.
PCR: Essential Techniques, (1996) Burke, J.F., ed., John Wiley & Sons, Ltd.
RNA
RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Second Edition, Farrell, R.E., Jr., ed., Academic Press, NY.
Acknowledgments
The author thanks Allan I. Basbaum for contributing to this story. Dr. Basbaum is in the Department of Anatomy, W.M. Keck Foundation Center for Integrative Neuroscience, and Department of Pediatrics, University of California, San Francisco, and is a contributing editor to the Journal of Neuroscience.
(b)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
(c)U.S. Pat. No. 4,766,072.
(d)Licensed under one or both of U.S. Pat. No. 5,487,993 and European Pat. No. 0 550 693.
pGEM is a registered trademark of Promega Corporation.
GenBank is a registered trademark of the U.S. Department of Health and Human Services.
All photos by Neal Cosby and property of Promega Corporation.
©1999 Promega Corporation. All rights reserved.
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