This review by Freeman, Walker, and Vrana discusses the challenges and potential of quantitative reverse transcription PCR (QRT-PCR) for analyzing RNA. The authors emphasize the importance of understanding the technical aspects of QRT-PCR, including the mathematical models underlying the process, the choice of RNA standards (internal vs. external), and quantification strategies (competitive, noncompetitive, and kinetic amplification). They highlight the variability in RT and PCR efficiencies, which can lead to significant errors in quantification. The review also covers practical considerations such as RNA isolation, reaction phase, heteroduplex formation, and the limitations of absolute quantification. The authors recommend the use of homologous synthetic RNA standards to control for variability and suggest combining competitive and noncompetitive approaches for more accurate quantification. They conclude by discussing the future potential of QRT-PCR in various scientific and clinical applications, emphasizing the need for careful experimental design and validation.This review by Freeman, Walker, and Vrana discusses the challenges and potential of quantitative reverse transcription PCR (QRT-PCR) for analyzing RNA. The authors emphasize the importance of understanding the technical aspects of QRT-PCR, including the mathematical models underlying the process, the choice of RNA standards (internal vs. external), and quantification strategies (competitive, noncompetitive, and kinetic amplification). They highlight the variability in RT and PCR efficiencies, which can lead to significant errors in quantification. The review also covers practical considerations such as RNA isolation, reaction phase, heteroduplex formation, and the limitations of absolute quantification. The authors recommend the use of homologous synthetic RNA standards to control for variability and suggest combining competitive and noncompetitive approaches for more accurate quantification. They conclude by discussing the future potential of QRT-PCR in various scientific and clinical applications, emphasizing the need for careful experimental design and validation.