An Example of ISH Based Target Validation

Background:  In vitro studies had shown that molecule y could stimulate proliferation of peripheral blood lymphocytes and increase cytotoxicity of NK cells. In the absence of a suitable antibody, a RNA probe was generated for fast ISH tissue profiling. The purpose of the ISH  analysis was to determine the origin of molecule y and to identify any disease processes that might be associated with the molecule.

 Procedures

 Results

Picture

 

 

 

Probe production

 

 

In vitro transcription produced high quality RNA probe (1 - sense template; 2- anti-sense template; 3 - sense RNA probe; 4 - anti-sense RNA probe).

 

 

 

ISH tissue profiling was performed in standard normal tissue and caner arrays, and our inflammatory pathology model.

 

 

ISH  tissue profiling showed a unique distribution of molecule y in normal tissues (blue bars) and increased levels (of the molecule) in a wide range of inflammatory (red bars) and cancerous (yellow bars) conditions

 

 

 

ISH tissue profiling of molecule y in normal tissue arrays.

 

Picture shows high expression of molecule y (black-brown staining indicated by blue arrows) in small intestine Paneth cells that are thought to secret molecules for regulation of microbial flora in the gut.

 

 

 

ISH tissue analysis of molecule y in inflammatory pathology model.

 

The right picture shows high expression level of molecule y in the infiltrating lymphocytes (black staining indicated by red arrows) of a case of inflammatory bowl disease.

 

 

 

ISH tissue analysis of molecule y in inflammatory pathology model.

 

The right picture shows high level of molecule y in the regenerating epithelial cells (black staining indicated by red arrows) in a case of chronic thyroiditis.

 

 

 

ISH tissue profiling of molecule y in cancer tissue arrays.

 

The right picture shows high level of molecule y in the infiltrating  lymphocytes (black staining indicated by red arrows) and mild level in the cancer cells (right lower part) in a case of colon cancer.

 

 

 

Summary and Conclusion

In normal tissues, molecule y was detected in Paneth cells of the small intestine, epithelial cells of the prostate and germ cells of the testes. Increased levels of the molecule were observed in a wide range of inflammatory and cancerous conditions including lymphocytes of the reactive lymph node/tonsil, crohn disease, ulcerative colitis, regenerating epithelial cells of chronic thyroiditis, and infiltrating lymphocytes and tumor cells of many tumor types. The increased levels demonstrated in these conditions indicate that the expression of molecule y may be associated with inflammation and general cell stress.

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