Project description:miRNAs are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time (RT)-PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA fluorescence in situ hybridization (FISH). For proof-of-concept, we differentiated two skin tumors, namely Basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified tumor-specific miRNAs (miR-205 and miR-375) in BCC and MCC respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and rRNA retention using model compounds and HPLC analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using pre-defined cut-off values; all were correctly identified in blinded analysis. We established a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues
Project description:Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a new mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We have developed the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We used the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which revealed that the stem cell gene, Sox2, is not essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.
Project description:miRNAs are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time (RT)-PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA fluorescence in situ hybridization (FISH). For proof-of-concept, we differentiated two skin tumors, namely Basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified tumor-specific miRNAs (miR-205 and miR-375) in BCC and MCC respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and rRNA retention using model compounds and HPLC analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using pre-defined cut-off values; all were correctly identified in blinded analysis. We established a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues 6 MCC samples 2 normal skin samples were profiled with Agilent miRNA array platform