Several methods have been developed for the detection of miRNAs, however, few allow the simultaneous detection of multiple miRNAs. To overcome this analytical deficiency, the Richert group at the University of Stuttgart has recently developed an ingenious method to selectively detect miRNAs on microarrays without interference from endogenous pre-mRNAs, mRNAs and other RNA species. In this method, a short oligonucleotide containing 3'-amino-dT and a 5' reporter molecule is chemically ligated to the microRNA in a one-step procedure by in situ activation of the microRNA. This is specifically achieved by taking advantage of the fact that miRNAs, unlike other RNAs, are 5'-phosphorylated. The reaction is template-directed (and thus sequence specific) and can be performed together with enzymatic 3'-extension/labelling, either in solution or on a support. The short DNA labelling strand may feature one of a variety of different labels, such as a biotin group or a fluorophore.
Coupling: No changes needed from standard method recommended by synthesizer manufacturer.
Deprotection: Deprotect with ammonium hydroxide for 17 hours at 55°C. Not compatible with AMA deprotection.
REFERENCES - MicroRNA LABELLING|(1) H. Vogel, and C. Richert, ChemBioChem, 2012, 13, 1474-82.|(2) R. Eisenhuth, and C. Richert, Journal of Organic Chemistry, 2008, 74, 26- 37.|(3) E. Kervio, A. Hochgesand, U.E. Steiner, and C. Richert, Proc Natl Acad Sci U S A, 2010, 107, 12074-9.