title: In situ detection of mature microRNAs by labeled extension on ultramer templates.
abstract: We describe a new method for the in situ detection of a mature microRNA (miRNA) in formalin-fixed, paraffin-embedded tissues. The method involves the labeled extension of miRNA hybridized to an approximately 100-nucleotide-long ultramer template containing the complementary sequence of the miRNA at its 3' terminus. Pretreatment of the tissue involves incubation with protease to expose the genomic DNA to DNase digestion, thereby eliminating the ultramer-independent DNA synthesis process inherent in paraffin-embedded tissue. By direct comparison with real-time reverse transcriptase (RT)-PCR, RT in situ PCR, and standard in situ hybridization using a locked nucleic acid (LNA) probe, it was evident that the ultramer extension method detects only the mature miRNA, is easier to optimize, results generally in a stronger signal, and is much less expensive than the LNA probe method currently used.
authors.affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210, USA. gnuovomd@pol.net
authors.forename: Gerard
authors.initials: G
authors.lastname: Nuovo
authors.forename: Eun Joo
authors.initials: EJ
authors.lastname: Lee
authors.forename: Sean
authors.initials: S
authors.lastname: Lawler
authors.forename: Jakub
authors.initials: J
authors.lastname: Godlewski
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmittgen