Columbia University Medical Center
Center for Radiological Research

NIH Program Project on
Radiation Bystander Effects: Mechanism
PO1-CA 49062-23

Technical Core

Core Leader: David J. Brenner

 

There are four main Core functions for this Technical Core:

  1. Provide consistent and rigorous statistical support for experimental design (power calculations) and data analyses.
  2. 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.
  3. Provide expertise in the design and fabrication of special irradiation fixtures through the Design and Instrument Shop of the Center for Radiological Research.
  4. Obtain and interpret transcriptomic and proteomic data.
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.
  • The 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 (0.5┬Ám) 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.

For detail, please refer RARAF homepage here!

 

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.

Image. General gene expression analysis workflow
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.


Personnel:


Dr. David Brenner, Director of Core

Dr. Sally Amundson, Co-investigator
Dr. Gerhard Randers-Pehrson, Chief Physicist
Gary Johnson, Director of Instrument Shop

 

New Links:
Design and Instrument Shop
RNA-Seq
Proteomics