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Gone FISHing for Cancer microRNAs

05/29/2013
Sarah C.P. Williams

For the first time, scientists have successfully applied fluorescent in situ hybridization (FISH) to tumor-specific microRNA markers. Learn how...


By fine-tuning the design of fluorescent probes and RNA fixation techniques, researchers have developed a way to detect and quantify microRNA molecules in tumor biopsy samples. The new fluorescence in situ hybridization (FISH) method allows the identification of tumor types that contain unique combinations of microRNAs. And unlike previous methods, the approach allows the simultaneous detection of multiple microRNAs while leaving the biopsy sample intact.

Merged images depict BCC in yellow and MCC in green, enabling rapid tumor identification. Source: Journal of Clinical Investigations




“People had developed RNA FISH methods before, but it had always been full of problems,” said Thomas Tuschl, a researcher at The Rockefeller University and Howard Hughes Medical Institute and author of a paper published in Journal of Clinical Investigations describing the new method (1). “And I think this paper is a new foundation for showing that a lot of preconceptions that were in the field had been wrong, and there were just no chemists getting into this field and really optimizing the methods.”

In FISH, fluorescent tags are designed to bind to specific RNA or DNA sequences, producing fluorescence that can be detected through a microscope. Since many cancers contain unique microRNAs or unique combinations of microRNAs, the detection of these molecules could be useful for cancer diagnosis. Applying FISH to tumor microRNAs has been slow-going though. Scientists have generally obtained low levels of fluorescence from tissue samples that are formalin-fixed and paraffin-embedded, the standard method for storing histological samples. The assumption has been that RNA denaturation was compromising the results.

But after measuring the overall ribosomal RNA in samples before and after their experiments, Tuschl’s team discovered that the weak results weren’t due to RNA degradation. Instead, the microRNAs weren’t being fixed properly, and the probe wasn’t sufficiently optimized.

So Tuschl's Rockefeller colleague Pavol Cekan led an effort to engineer and test different versions of the probe. He found that the optimal design included a longer linker attached to a hapten, producing a probe that can be amplified to get a large fluorescent signal. “If you use the commercial products that a number of companies sell, they don’t work for this because the linkers are too short,” explained Tuschl.

To evaluate Cekan’s probes, Neil Renwick—a Rockfeller researcher who was a pathologist by training—chose to test whether the new method would allow him to differentiate between two skin cancers that can have similar histology: basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC). “Each cancer has one unique microRNA, which makes it easy to study,” he said.

Using only the levels of two key microRNAs from 5 BCC tumors and 13 MCC tumors, the team correctly identified the tumor type of each sample.

And now Tuschl and Cekan have already increased the number of RNAs they can detect at once. “We have now measured seven RNAs plus DAPI in one experiment—eight fluorophores at once,” said Tuschl. “And this is just the beginning. Our controls done with the probe for ribosomal RNA point to the possibility that this will translate to other RNAs as well.”

References

1. Renwick, N., P. Cekan, P.A. Masry, S.E. McGeary, J.B. Miller, et al. 2013. Multicolor microRNA FISH effectively differentiates tumor types. The Journal of Clinical Investigation, 123:6 2694-2700.

Keywords:  microscopy cancer microRNA