In Situ Hybridization - Process

Process

For hybridization histochemistry, sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. As noted above, the probe is either a labeled complementary DNA or, now most commonly, a complementary RNA (riboprobe). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e. only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (e.g., digoxigenin) is localized and quantified in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

An alternative technology, branched DNA assay, can be used for RNA (mRNA, lncRNA and miRNA ) in situ hybridization assays with single molecule sensitivity without the use of radioactivity. This approach (e.g., ViewRNA assays) can be used to visualize up to four targets in one assay and it uses patented probe design and bDNA signal amplification to generate sensitive and specific signals. Samples (cells, tissues and CTCs) are fixed, then treated to allow RNA target accessibility (RNA un-masking). Target-specific probes hybridize to each target RNA. Subsequent signal amplification is predicated on specific hybridization of adjacent probes (individual oligos that bind side by side on RNA targets). A typical target-specific probe will contain 40 oligonucleotides, resulting in 20 oligo pairs for detection of mRNA and lncRNA and 2 oligos or a single pair for miRNA detection. Signal amplification is achieved via a series of sequential hybridization steps. A pre-amplifier molecule hybridizes to each oligo pair on the target-specific RNA, then multiple amplifier molecules hybridize to each pre-amplifier. Next, multiple label probe oligonucleotides (conjugated to alkaline phosphate or directly to fluorophores) hybridize to each amplifier molecule. A fully assembled signal amplification structure “Tree” has 400 binding sites for the label probes. When all target-specific probes bind to the target mRNA transcript, an 8,000 fold signal amplification occurs for that one transcript. Separate but compatible signal amplification systems enable the multiplex assays. The signal can be visualized using a fluorescence or brightfield microscope.