Induction of gene expression and subsequent detection by FISH-Flow. c-fos expression is induced in activated cells, which are readily identified by FISH-Flow. GFP: negative control, GAPDH: positive control.
Invention Summary:
Many immune responses to pathological conditions involve the activation of T-cells by specific antigens. The interactions between the antigens and the T-cell receptors (TCRs) lead to intracellular signaling cascades, which trigger changes in gene expression. These changes are typically assessed by protein presence, either at the cell surface or secreted, for clinical diagnostic purposes. However, there are drawbacks to this approach including: 1) approaches that detect proteins do not accurately reflect the kinetics of gene expression, 2) well-characterized antibodies for many targets are not available, and 3) secreted markers (e.g. cytokines) cannot be detected with antibodies.
Researchers at Rutgers have developed an assay combining the analytical power of flow cytometry and fluorescence in situ hybridization (FISH-Flow) for high-throughput identification of activated T-cells. Gene expression at the mRNA level and expression of cell surface markers can be assayed simultaneously in activated T-cell populations using this technique. The use of nucleic acid probes provides exceptional target flexibility while maintaining exquisite sensitivity; fewer than 10 mRNA molecules per cell can be detected by FISH-Flow. After being analyzed by flow cytometry, cell populations can be sorted for additional downstream analysis and multiparametric phenotyping. A semi-automated protocol has been developed which allows for preparation and analysis of up to 40 samples simultaneously. RNA targets can be selected from RNA-seq data, and probes can be prepared for any RNA.
Market Applications:
Suitable for use in diagnostic and/or research applications requiring any of the following:
- Rapid identification of activated T cells and/or identification of low-abundance T cell populations, such as antigen-specific memory cells
- Identification of any cell type or any tissue responding to a specific stimulation
- Multiparametric immunophenotyping
- Concurrent detection of mRNA targets and cell surface markers
Advantages:
- Enhanced temporal resolution of dynamic gene expression events
- Eliminates the need for protein-secretion inhibitors when detecting secreted proteins such as cytokines
- Nucleic acid probes provide greater target flexibility (vs. antibody probes)
- Semiautomated protocol reduces cell loss, operator time, and interoperator variability
Intellectual Property & Development Status: Patent issued in US (10900072) and EU (EP2867374). Available for licensing and/or research collaboration. Please contact alex.turo@rutgers.edu.
Publication: Y. et al. (2015) Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry. J. Immunol. DOI: 10.4049/jimmunol.1401515.
