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February 2, 2009 In situ hybridization (ISH) is great to tell how much miRNA is expressed where. But it’s not so easy to get a signal from low-abundance miRNAs in formalin-fixed tissue. Formaldehyde cross-links RNA to proteins, diminishing the ability of probes to recognize and bind to the RNA, and can modify some of the bases as well. Heating things up reverses these effects to some extent, but then the miRNA tends to diffuse away, leaving nothing to profile.
Researchers in Thomas Tuschl’s Lab at Rockefeller used a combination of approaches to solve the problem. After formalin fixation -- but prior to incubation with the probe -- they treat the tissue with EDC, a water-soluble fixative that irreversibly links the RNA’s 5’ phosphate to protein side chains. They then probe with linked nucleic acid (LNA) oligos, which put a hybridization smack-down on miRNAs due to the higher binding affinity these mutant oligos exhibit than those synthesized from DNA alone. The result is a stronger, more reliable signal from even rarer miRNAs. Check out the details in Nature Methods, Jan 2009
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