Probes for KRT19

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development.

Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

Cellular senescence is a mechanism that provides an irreversible barrier to cell cycle progression to prevent undesired proliferation. However, under pathological circumstances, senescence can adversely affect organ function, viability and regeneration. We have developed a mouse model of biliary senescence, based on the conditional deletion of Mdm2 in bile ducts under the control of the Krt19 promoter, that exhibits features of biliary disease. Here we report that senescent cholangiocytes induce profound alterations in the cellular and signalling microenvironment, with recruitment of myofibroblasts and macrophages causing collagen deposition, TGFβ production and induction of senescence in surrounding cholangiocytes and hepatocytes. Finally, we study how inhibition of TGFβ-signalling disrupts the transmission of senescence and restores liver function. We identify cellular senescence as a detrimental mechanism in the development of biliary injury. Our results identify TGFβ as a potential therapeutic target to limit senescence-dependent aggravation in human cholangiopathies.