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FISHing for the right probe

Fluorescence in situ hybridization (FISH) is a cytogenetic technique used to detect the absence/presence and location of specific DNA/RNA sequences. This technique allows the microscopic visualization of abnormalities such as chromosomal deletion, amplification and translocation/inversion. Pathologists can select a specific area of a tumor to be examined by FISH and directly correlate FISH results obtained under fluorescence or confocal microscopy and tumor morphology observed under conventional light microscopy. FISH is currently used for prenatal diagnosis as well as a diagnostic and prognostic tool for various cancer types. Successful FISH requires optimization of several elements including tissue fixation and permeabilization, design of the fluorescently-labeled target-specific nucleic acid probes, and their annealing and detection within the tissue of interest. Despite the development and the availability of different RNA FISH methods, specialists struggled with low levels of fluorescence when using formalin-fixed and paraffin-embedded (FFPE) tissue samples. Recently, Dr. Renwick and colleagues from the Rockefeller University demonstrated that the poor quality of the results was not due to RNA denaturation occurring during fixation and embedding of the tissue sample but to the lack of optimization done on the probes. Refined and enhanced fluorescence signals were obtained by increasing probe-hapten linker length and shortening probes to minimize ribosomal RNA sequence complementarity. The researchers showed the utility of this optimization when detecting novel microRNAs (miRNAs) that could differentiate between two skin cancers, miR-205 in basal cell carcinoma (BCC) and miR-375 in Merkel cell carcinoma (MCC). As a proof of concept, they designed probes against these two miRNAs, normalized the signals obtained against directly detectable reference ribosomal RNA signals and successfully identified tumors in blinded analysis. This technique should be widely applicable to all miRNAs in any FFPE tissue. However, it remains to be seen whether this technique works in a larger sample collection before implementing it as a routine test in clinical diagnosis laboratory.

Enzo Life Sciences offers a comprehensive portfolio for FISH and ISH analysis including labeling, typing and detection assays, deoxynucleotides, dideoxynucleotides and ribonucleotides, some of which are described below:

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