This paper presents a novel method for real-time quantification of microRNAs (miRNAs) using stem-loop reverse transcription (RT) followed by TaqMan PCR analysis. The stem-loop RT primers are more efficient and specific than conventional primers, and the TaqMan miRNA assays are highly specific for mature miRNAs, discriminating between related miRNAs that differ by as little as one nucleotide. The method is highly sensitive, achieving precise quantification with as little as 25 pg of total RNA for most miRNAs. It can be used to analyze single cells without nucleic acid purification and has a dynamic range of seven orders of magnitude. The method was validated using mouse tissues and cell lines, demonstrating its ability to profile miRNA expression across different tissues and cell types. The authors also compared the performance of stem-loop and linear RT primers, finding that stem-loop primers offer better specificity and sensitivity. This method provides a practical solution for the sensitive and specific profiling of miRNAs, which is crucial for identifying biomarkers specific to tissues or diseases.This paper presents a novel method for real-time quantification of microRNAs (miRNAs) using stem-loop reverse transcription (RT) followed by TaqMan PCR analysis. The stem-loop RT primers are more efficient and specific than conventional primers, and the TaqMan miRNA assays are highly specific for mature miRNAs, discriminating between related miRNAs that differ by as little as one nucleotide. The method is highly sensitive, achieving precise quantification with as little as 25 pg of total RNA for most miRNAs. It can be used to analyze single cells without nucleic acid purification and has a dynamic range of seven orders of magnitude. The method was validated using mouse tissues and cell lines, demonstrating its ability to profile miRNA expression across different tissues and cell types. The authors also compared the performance of stem-loop and linear RT primers, finding that stem-loop primers offer better specificity and sensitivity. This method provides a practical solution for the sensitive and specific profiling of miRNAs, which is crucial for identifying biomarkers specific to tissues or diseases.