Collection OverviewIn Situ Hybridization (ISH) provides a powerful tool for localized detection of RNA/DNA sequences, widely applied in both research and clinical contexts. This technique takes advantage of nucleic acid probes that are complementary to target RNA or DNA sequences, in order to localize the target sequences on chromosomes, in tissues, cells, organs or organisms. RNA ISH can be used to identify deregulated expression of mRNA, lncRNA, snRNA or miRNA transcripts, which can manifest as expression mis-lo… Show Morecalization or expression levels’ changes. DNA ISH is useful for distinguishing gene alleles, identifying chromosomal abnormalities/rearrangements, as well as detecting viral, bacterial or fungal genes commonly seen in infectious diseases.
This Methods Collection aims at creating a comprehensive collection of the most current protocols of RNA/DNA ISH; understanding the applicability, advantages and limitations of this technique will allow researchers to utilize it to its full potential. Examples of ISH protocols that can be included in this collection are: chromogenic or fluorescent ISH, whole mount, section or cell ISH, as well as assays that combine ISH with other techniques such as protein immunohistochemistry or PCR amplification. Show Less
Next generation RNAscope/DNAscope in situ hybridization applications in HIV/SIV research
In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. ISH has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach (termed RNAscope) was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve si… Show Morengle-molecule visualization while preserving tissue morphology. This next-generation of ISH is based on an approach similar to branched PCR, but performed in situ and is more facile, sensitive and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has become the leading experts in developing and applying the RNAscope ISH platform to the detection of HIV and SIV vRNA and/or vDNA+ cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and tips to all individuals interested in using RNAscope ISH in their research