Leader: David J.
There are four main Core functions for this Technical Core:
The Radiological Research Accelerator
Facility (RARAF) is dedicated to providing user-friendly radiation
sources and bio-labs for research in biology, radiation biology,
and radiation physics. RARAF features a single-cell/single-particle
microbeam irradiator which, together with integrated techniques
for imaging cellular, sub-cellular, or small-tissue-volume targets,
allow precisely defined quantities of damage to be induced at
precisely defined locations.
- Provide consistent and rigorous statistical support for experimental design (power calculations) and data analyses.
- Provide support for the Program Project related operations of the accelerator-based single-cell / single-particle microbeam [link: http://www.raraf.org/microbeams.html] and track-segment [link: http://www.raraf.org/tracksegment.html] charged-particle irradiation facilities, and dosimetry support for all irradiations.
- Provide expertise in the design and fabrication of special irradiation fixtures through the Design and Instrument Shop of the Center for Radiological Research.
- Obtain and interpret transcriptomic and proteomic data.
RARAF microbeam has been used to target sub-cellular structures
such as the cell nucleus for years. Now that a focused charged-particle
beam with sub-micron diameter (0.5µm) is routinely available,
additional targets within cellular systems are accessible.
For instance, preliminary radiation experiments that target
mitochondria have been conducted on small airway epithelial
cells. In these experiments, the RARAF multiphoton microscope
was used to image GFP-tagged mitochondria sites and position
them over the ion beam for irradiation. The figure shows a
multiphoton microscope image of mitochondria (green) targets
within human small airway epithelial cells. The crosshairs
(red) mark the center of the image, which coincides with the
position of the ion beam. For this image and for post-irradiation
time-lapse images of mitochondria behavior, Hoechst was used
to counter stain the nuclei.
- Multiphoton microscopy, a laser-based, 3D imaging technique
is integrated into the Microbeam II endstation at RARAF. The
multiphoton microscope was custom-designed around the Nikon
Eclipse E600-FN research fluorescence microscope at the endstation
and is intended for imaging cell dynamics in tissue and cell-culture
samples following irradiation. Intended for detecting and
observing short-term molecular kinetics of radiation responses
in living tissue and in cell-culture samples, this multiphoton
microscope is the first of its kind to be assembled onto a
microbeam cell-irradiation platform.
- The multiphoton microscope allows users of the RARAF microbeam
to perform in vivo microbeam studies. Researchers
at RARAF are now irradiating small, living organisms, such
as the C. elegans nematode, the Medaka and Zebrafish
embryos, and the hairless mouse ear. In vitro tissue
systems are also available.
- Additionally, the single-cell/single-particle microbeam
irradiator is constantly being updated with new technologies
designed to provide new microbeam sources, single cell manipulation
technologies, enhanced imaging, higher throughput in support
of the Program Project grant for irradiation of small-animal
systems to RARAF.
detail, please refer RARAF homepage
General gene expression analysis workflow
RNA Quality: Before any microarray hybridization can be undertaken, it is necessary to confirm that the RNA to be labeled is of acceptable quality. We use the Agilent Bioanalyzer to accomplish this step.
Amplification and labeling: Small amounts of RNA are routinely amplified prior to labeling and hybridization. For this step we use the Agilent system for direct labeling of amplified cRNA.
Microarray hybridizations will be conducted using human or mouse Agilent whole genome microarrays as appropriate and standard hybridization and washing conditions.
Scanning: Microarrays are scanned using the Agilent DNA Microarray Scanner and its Feature Extraction Software, which is compatible with industry standard analysis applications.
Downstream Data Analysis: We continue to use previously developed tools for data analysis, while also exploring opportunities for development and application of more sophisticated and specific tools through collaborations.
Future Directions: We are beginning to incorporate RNA-Seq and proteomic analyses into our studies through the Shared Resources of the Herbert Irving Comprehensive Cancer Center, where these offer additional power for achieving our aims.
Dr. David Brenner, Director of Core
Dr. Sally Amundson, Co-investigator
Dr. Gerhard Randers-Pehrson, Chief Physicist
Gary Johnson, Director of Instrument Shop
Design and Instrument Shop