With the Odyssey scanner's dynamic range we can reliably measure modest changes — as little as 1.5 fold — at the protein level.
Dr. Stefan Hüttelmaier
Director of Core Facility Imaging
Martin Luther University
Dr. Stefan Hüttelmaier, Director of Core Facility Imaging at Martin Luther University, studies regulation and post-transcriptional control of RNA viral proteins and micro-RNAs.
Dr. Hüttelmaier has been studying RNA molecules since his post-doc at the Albert Einstein College of Medicine in New York. There he worked in the Singer lab, which studies RNA localization issues. Hüttelmaier is now at the Institute of Molecular Medicine in the Division for Molecular Cell Biology in Halle, Germany, where his main interest is the cellular function of protein-RNA complexes.
According to Hüttelmaier, protein-RNA interactions are a major area of interest in his field. Historically, protein-RNA interactions have been analyzed by radiolabeling and autoradiography. However, when Dr. Hüttelmaier opened his lab in Germany, none of his 20 students were interested in using radioactive assays on a daily basis. Hüttelmaier clarifies, "so I said okay, then let's have a look at whether we can do this by fluorescently labeling the RNAs."
Rethinking Conventional Research Techniques
Dr. Hüttelmaier's inspiration to try analysis with fluorescent labels went against traditional methods. "Most elder scientists would recommend using radioactive RNA, otherwise it's not really reliable." Nevertheless Dr. Hüttelmaier purchased an Odyssey® Imager and incorporated fluorescent imaging technology into his RNA research successfully.
"There are other advantages aside from the minimized risk for the operator's health," says Hüttelmaier. "The main assay we use it for is Western blotting: It is more reliable, has a higher dynamic range, and of course is much faster and much cheaper than doing this by conventional chemiluminescent methodology. We found that with the Odyssey scanner's dynamic range we can reliably measure modest changes — as little as 1.5 fold — at the protein level."
The ability of the Odyssey Imager to detect two targets simultaneously is critical for its reliability and reproducibility. Hüttelmaier uses this technology to characterize and quantify bands with similar molecular weight. He explains, "Isotope labeling doesn't allow differentiating between two RNAs of the same size, right? So the idea was to use two similar sized RNAs that were tagged with fluorophore-conjugated nucleotides emitting at either 700 or 800 nanometers. And herewith, we were able to show different complexes of similar size by the two colors and even show that there is a specific redistribution."
A Reliable, User-Friendly Technology
The Odyssey is an essential piece of equipment in our lab. It's running all day long here.
Dr. Hüttelmaier and his colleagues can now boast several papers using near-infrared Western blots, and fluorescent RNA protein assays (i.e. filter binding assays, and electrophoretic mobility shift assays (EMSAs)) for analysis of protein-RNA complexes and RNA cross-linking to proteins. "We have used infrared dye conjugated nucleotides to randomly label RNAs by in vitro transcription. This allows detection via the Odyssey imager in the low femtomolar range and essentially enables protein-RNA interaction studies."
In addition to getting reliable results at a cost savings and time savings with a method that is safe, Dr. Hüttelmaier appreciates how easy it is to train new lab personnel to run the Odyssey Imager. He says, "The Odyssey is an essential piece of equipment in our lab. It's running all day long here." His lab continues to develop additional fluorescent RNA assays which he hopes to publish in the near future.
Dr. Hüttelmaier's experience with the adaption of hazardous radioactive assays to infrared assays for the Odyssey Imager is just one of the reasons we are proud to call him an "Odyssey Expert."
Publications resulting from work on the Odyssey
- Berndt H, Harnisch C, Rammelt C, Stöhr N, Zirkel A, Dohm JC, Himmelbauer H, Tavanez JP, Hüttelmaier S, Wahle E. (May 2012) Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming RNA 18(5): 958-972.
- Stöhr N, Köhn M, Lederer M, Glaβ M, Reinke C, Singer RH, Hüttelmaier S. (Jan 2012) IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling Genes Dev. 26(2): 176-189.
- Braun J, Hoang-Vu C, Dralle H, Hüttelmaier S. (Jul 2010) Down-regulation of microRNAs directs EMT and invasive potential of Anaplastic Thyroid Carcinomas Oncogene 29(29): 4237-4244.
- Köhn M, Lederer M, Wächter K., Hüttelmaier S. (Jul 2010) Near infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the non-coding Y3-RNA RNA 16(7): 1420-1428. Weidensdorfer D., Stöhr N., Baude A, Lederer M, Köhn M, Schierhorn A, Buchmeier S, Wahle E, Hüttelmaier S. (Jan 2009) Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs RNA 15(1): 104-115.
- Dr. Geir Bjørkøy
University College of Sør-Trøndelag and CEMIR Centre of Excellence, NTNU
- Dr. Wendy Bollag
Georgia Regents University, Charlie Norwood VA Medical Center
- Dr. Jeremy Chambers
Florida International University, Miami, FL
- Dr. Paul Digard
University of Cambridge, UK
- Dr. Jane Gray
Cancer Research Institute, Cambridge, UK
- Manuela Gustafsson
Karolinska Institute's Department of Laboratory Medicine
- Dr. Oliver Hantschel
- Dr. Stefan Hüttelmaier
Martin Luther University
- Dr. Jelena Janjic
- Dr. Andrea Kasinski
- Dr. Sue Katz
Rogers State University
- Dr. Lisa Keyes
University of Florida College of Medicine
- Dr. Stanislav Kholmanskikh
Weill Cornell Medical College
- Dr. Patrick Kiely
University College Cork, Ireland
- Dr. Andreas von Knethen
Johann Wolfgang Goethe-University
- Dr. Michael Lee
University of Massachusetts Medical School
- Dr. Robert Lewis
University of Nebraska
- Dr. H. Charles Manning
- Dr. Debra Martin
Saint Mary's University of Minnesota
- Dr. Kevin Morano
The University of Texas Graduate School of Biomedical Sciences at Houston
- Dr. Rick Page
Miami University in Oxford, OH
- Dr. Nicolai Peschel
Universität Würzburg Department of Neurobiology & Genetics
- Dr. Wendy Picking
Oklahoma State University
- Dr. Martin Pomper
Johns Hopkins Medical School
- Dr. Vikash Reebye
Imperial College London, UK
- Dr. Rosemary Schuh
University of Maryland School of Medicine