More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.