EXTassays, toward maturity of RNA reporters

ResearchBlogging.orgI read with some interest a recent Nature Methods paper appeared this January. Anna Botvinnik and colleagues from Max Planck Institute, conceived a new reporter system able to measure receptor activation (receptor dimerization), downstream signaling (adapter recruitment) and subsequnent cis-regulatory responsive elements transactivation efficacies by...

...no, you don't need a 64-milion new-generation machine, you need Trizol!

As I reviewed in my first 2010 post, there is a trend to develop multiple reporter into a library ready to transfect. In this paper, the authors just coupled each reporter with a unique expressed oligonucleotide tag, transfected the library, isolated the reporter mRNAs as a pool and analyzed it by hybridization to microarray. In brief, this is EXTassay. Wondering about transfection efficiency? Owing to its small scale, plasmid DNA was not denaturated by Trizol and was localized in the acqueous phase (they said), so it can be isolated and hybridized to serve as calibrator.

The trick to probe receptor dimerization relies in the genetic manipulation of the receptor gene, and it is quite similar to the Tango assay. Other than being solely a receptor, the protein in exam is coupled in frame with partial TEV recombinase and a GV transcription factor. Upon receptor dimerization (activation) follows reconstitution of a TEV protease and cleavage of GV, which goes in the nucleus where it can find a corresponding GV-reporter.

Despite I'm still uncomfortable with the idea to interrogate receptor activity by receptor transfection, I start feeling RNA-reporters being on the road toward maturity, and I admit: probably I'have been too much severe with Tango assays in January 2008. I should think at this when planning my future experiments, there is more other than luciferase in 2010.

---/ Citation /--- --- --- ---
Botvinnik, A., Wichert, S., Fischer, T., & Rossner, M. (2010). Integrated analysis of receptor activation and downstream signaling with EXTassays Nature Methods, 7 (1), 74-80 DOI: 10.1038/nmeth.1407