A novel method for real-time quantification of microRNAs (miRNAs) using stem-loop RT followed by TaqMan PCR analysis is described. This method uses stem-loop RT primers, which are more efficient and specific than conventional ones. TaqMan miRNA assays are specific for mature miRNAs and can distinguish between related miRNAs differing by as little as one nucleotide. They are not affected by genomic DNA contamination. The method allows for precise quantification of miRNAs with as little as 25 pg of total RNA. It enables direct analysis of single cells without nucleic acid purification and has a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method allows for fast, accurate, and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for quantifying other small RNA molecules such as siRNAs. The concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency. The method involves stem-loop RT followed by real-time PCR. RNA samples from various mouse tissues and cells were used. The method was validated using synthetic miRNAs and showed excellent linearity between the log of target input and Ct values, demonstrating a dynamic range of at least 7 logs. The method was also tested with heat-treated cells and showed good results. The method was compared with solution-based northern hybridization analysis and showed better specificity and sensitivity. The stem-loop RT primers provided better RT efficiency and specificity than linear ones. The method is highly specific for mature miRNAs and can distinguish between mature miRNAs and their precursors. The method is suitable for both miRNA and precursor quantification. The method is sensitive and specific, allowing for accurate quantification of miRNAs in various tissues and cells. The method has potential applications in identifying miRNA biomarkers and understanding miRNA regulation in various biological processes.A novel method for real-time quantification of microRNAs (miRNAs) using stem-loop RT followed by TaqMan PCR analysis is described. This method uses stem-loop RT primers, which are more efficient and specific than conventional ones. TaqMan miRNA assays are specific for mature miRNAs and can distinguish between related miRNAs differing by as little as one nucleotide. They are not affected by genomic DNA contamination. The method allows for precise quantification of miRNAs with as little as 25 pg of total RNA. It enables direct analysis of single cells without nucleic acid purification and has a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method allows for fast, accurate, and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for quantifying other small RNA molecules such as siRNAs. The concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency. The method involves stem-loop RT followed by real-time PCR. RNA samples from various mouse tissues and cells were used. The method was validated using synthetic miRNAs and showed excellent linearity between the log of target input and Ct values, demonstrating a dynamic range of at least 7 logs. The method was also tested with heat-treated cells and showed good results. The method was compared with solution-based northern hybridization analysis and showed better specificity and sensitivity. The stem-loop RT primers provided better RT efficiency and specificity than linear ones. The method is highly specific for mature miRNAs and can distinguish between mature miRNAs and their precursors. The method is suitable for both miRNA and precursor quantification. The method is sensitive and specific, allowing for accurate quantification of miRNAs in various tissues and cells. The method has potential applications in identifying miRNA biomarkers and understanding miRNA regulation in various biological processes.