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Improved RNA detection technique enhances ability to monitor cellular messages

Scientists have improved an existing technique to detect RNA messages inside cells, making it faster and cheaper to simultaneously track the activity of multiple genes. Messenger RNAs (mRNAs) carry protein assembly instructions from genes to the cellular machinery where proteins are made. Technologies that track which mRNAs are in what cells at what time can be powerful, versatile tools. These tools can help researchers better understand the roles of the proteins the mRNAs encode, especially in processes where the activity of many genes changes at once, such as when healthy cells are disrupted by disease.

Researchers have now reported on improvements to one RNA detection method, called clampFISH. clampFISH uses a series of probes customized to stick to mRNAs encoding instructions for a specific protein of interest. A large molecular scaffold is then built over the mRNA, binding fluorescent dyes that amplify the relatively weak “signal” from a single mRNA so it can be detected by microscopy. A weakness of the original “clampFISH 1.0” procedure, however, was that it was relatively expensive and time-consuming, especially when detecting multiple different mRNAs at once.

To create clampFISH 2.0, the scientists modified the structure of the scaffold and streamlined the protocol to make it faster and cheaper. They verified that clampFISH 2.0 still accurately and precisely detected targeted mRNAs and that it could pinpoint an mRNA’s location within a cell. clampFISH 2.0 also generated a strong enough signal to allow imaging by lower-powered microscopes with wider fields of view, which allowed researchers to analyze cells faster. This feature was particularly useful in studying rare events. For example, scientists used clampFISH 2.0 in a very large tumor cell sample to measure the simultaneous activity of 10 genes, observing for the first time how those genes activate together in rare instances that can lead to drug resistance. Researchers also performed a similar analysis on preserved tumor tissue, such as would be generated by a cancer biopsy.

Overall, the streamlined clampFISH 2.0 procedure was shown to be a rapid and flexible tool to efficiently detect multiple mRNAs at once, allowing the study of research questions previously hindered by technical limitations.

Dardani I, Emert BL, Goyal Y,…Raj A. ClampFISH 2.0 enables rapid, scalable amplified RNA detection in situ. Nat Methods 19: 1403-1410, 2022.

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