Fluorescent In Situ Hybridization Detection of microRNAs in Newt Tissue Cryosections

Even though numerous protocols exist using in situ hybridization techniques to identify RNA targets within whole mount samples or tissue sections, a methodological finesse is still required in order to obtain specific labelling, free of high non-specific background staining. One parameter that is paramount to the specificity of staining is the in situ probe design. Large RNA molecules can be probed with long DNA oligonucleotide sequences that bind with high specificity to their target RNA, which have high melting temperatures and facilitate high stringency washing. However, small RNA targets, like 20–24 bp long microRNAs (miRs), require a probe that conveys high specificity with high melting temperatures, which are features sadly lacking from DNA oligonucleotide designed probes. Exiqon, a leading supplier of products for miR research, provide predesigned and custom made miRCURY LNA™ microRNA Detection Probes, which due to patented locked nucleic acid (LNA) technology, provide a high melting temperature probe that specifically binds to miRs of interest. To help facilitate the use of these probes for highly specific detection of miRs within tissue samples, an in situ hybridization protocol has been outlined for use on cardiac cryosections derived from red-spotted newts. Even though the protocol was originally designed to highlight the importance of miRs in a cardiac regeneration context, the in situ hybridization procedure described can also be adapted to alternativ...
Source: Springer protocols feed by Neuroscience - Category: Neuroscience Source Type: news