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Abstract Background MicroRNAs (miRNAs) are small non-coding RNAs (about 21 to 24 nucleotides in length) that effectively reduce the translation of their target mRNAs. Several studies have shown miRNAs to be differentially expressed in prostate cancer, many of which are found in fragile regions of chromosomes. Expression profiles of miRNAs can provide information to separate malignancies based upon stage, progression and prognosis. Here we describe research prototype assays that detect a number of miRNA sequences with high analytical sensitivity and specificity, including miR-21, miR-182, miR-221 and miR-222, which were identified through expression profiling experiments with prostate cancer specimens. The miRNAs were isolated, amplified and quantified using magnetic bead-based target capture and a modified form of Transcription-Mediated Amplification (TMA). Results Analytical sensitivity and specificity were demonstrated in model system experiments using synthetic mature microRNAs or in vitro miRNA hairpin precursor transcripts. Research prototype assays for miR-21, miR-182, miR-221 and miR-222 provided analytical sensitivities ranging from 50 to 500 copies of target per reaction in sample transport medium. Specific capture and detection of mature miR-221 from complex samples was demonstrated in total RNA isolated from human prostate cancer cell lines and xenografts. Conclusion Research prototype real-time TMA assays for microRNAs provide accurate and reproducible quantitation using 10 nanograms of input total RNA. These assays can also be used directly with tissue specimens, without the need for a preanalytic RNA isolation step, and thus provide a high-throughput method of microRNA profiling in clinical specimens.