Ligation in situ Hybridization for highly multiplexed RNA analysis of fixed tissue specimens

Unmet Need: Formalin fixed and paraffin embedded (FFPE) tissues are the most common form of patient specimens analyzed by pathologists. This standard practice of preserving clinical samples, however, dramatically damages the RNA content of these valuable specimens. RNA molecules provide important information about which genes are being expressed in a tissue, and whether or not there may be an ongoing infection. Current approaches to FFPE RNA analysis rely on RNA purification followed by reverse transcription (RT), processes that can reduce the sensitivity of downstream analyses and/or introduce unexpected artifacts. A more robust, sensitive and multiplexed analytical platform, which does not rely on RNA purification or reverse transcription, is therefore needed for the analysis of FFPE RNA in the research and clinical settings.
 
Technical Details: JHU inventors have developed a technique (Ligation In Situ Hybridization – LISH) to simultaneously detect and quantify the expression levels of 100's to 1,000’s of selected RNA targets, which is optimally suited for FFPE samples. LISH obviates the need for RNA extraction and reverse transcription. In this method, nucleic acid probe sets are hybridized adjacent to one another on their intended target RNA molecules, where they can be ligated enzymatically in situ. Ligated probe sets are then retrieved and can be PCR amplified using common primer sequences for downstream analysis, including qPCR, next generation DNA sequencing, and/or single molecule analysis. LISH demonstrates sensitivity equal to or greater than RT-qPCR. The method is compatible with many widely used histological stains and can be combined with laser capture microdissection for spatially resolved analyses. Primary perceived uses of LISH include gene expression analysis of tumors and their microenvironments, and the diagnosis of infections.
 
Patent Status: Pending
 
Stage of development/data: Pre-clinical. JHU is looking for partners to commercialize this technology.
 
Technology Category: Oncology, Infectious Disease, In Vitro Diagnostics, Diagnostics, Biomarkers
 
Publication:
https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkx471
Nucleic Acids Res. 2017 Aug 21;45(14):e128
 
 
 
 

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