Procedure for Molecular Profiling of Cells and Tissue Specimens

Principal Investigator: Xiaohu Gao

Understanding the pathological processes of diseases and the consequent clinical diagnostics is limited by lack of knowledge of predictive biomarkers that can enable discrimination between disease and normal function, different disease subtypes, and progression of the pathological process. Conventional biomedical techniques are limited in the number of biomarkers they can analyze simultaneously and are often unable to provide quantitative information due to the complexity and heterogeneity of the diseases. Multiplex detection requires unique assignment of biomarkers to corresponding detectable probes such as quantum dot (QD) probes. This can be achieved either through direct QD-antibody conjugation or unique nucleic acid encoding of each

The inventors have developed a novel hybrid 2-step immunofluorescence detection method, which allows biomarkers in biological samples to be assigned to corresponding fluorescent QD probes. During the first step of this hybrid method, biomarkers of interest are detected by binding to a specific primary Ab carrying unique DNA tags, which allows the DNA sequence code to be recognized in a second step. During the second step, QD-DNA probes can then be hybridized to the DNA tags that were generated in the first step and detected, thus overcoming limitations imposed by using primary and secondary Ab pairs. This hybrid immunofluorescence/Fluorescence In Situ Hybridization method with unique QD probes allows multiplex biomarker profiling in biological samples.

• The detection method can be utilized in experimental methods such as immunofluorescence, immunohistochemistry, and Western blotting for detecting multiple proteins at a time. 

• The invention may be applied in multiplex staining for molecular profiling of individual cells or cell populations.

• The hybrid immunofluorescence method is equally amenable for QD-based probes as well as conventional DNA-probes labeled with organic fluorophores. This makes this technique widely applicable to a large number of studies with different aims and parameters. 

• Multiplex detection has been hampered by limited availability of Primary Ab/Secondary Ab pairs. The hybrid IF/FISH procedure helps to navigate this limited availability.

• Due to the ability of the QDs and immunofluorescence method to detect multiple biomarkers, it can be used, for example, in diseases such as cancers that have multiple phenotypes within the same population. This is especially essential when some of the phenotypes are much rarer than others.

For more info, contact: Roï Eisenkot