Revisiting qRT-PCR for gene expression profiling in single cells toward diagnostics

Quantitative reverse-transcription PCR (qRT-qPCR) remains one of the most precise methods for measuring transcript abundance, yet its application at single-cell resolution poses challenges that differ fundamentally from bulk workflows. As interest in single-cell biology grows, there is a pressing need for methods capable of providing absolute, reproducible, and gene-specific measurements in individual cells. Single-cell qRT-qPCR (scRT-qPCR) holds strong potential to meet this need, offering true molecular quantification that complements the broader but less precise information obtained from single-cell RNA sequencing. However, its practical implementation has been hindered by deep chemical and biochemical conflicts among the reaction phases, by enzyme instability in nanoliter-scale environments, and by the absence of integrated systems that can support lysis, reverse transcription, and amplification within a single unified workflow. This review synthesizes the chemical foundations that distinguish single-cell reactions from bulk qRT-qPCR and explains why conventional protocols cannot simply be miniaturized. By examining the molecular origins of reaction incompatibility and the physical constraints imposed by extremely low input levels, we identify the key factors that limit current methods. We also evaluate the strengths and shortcomings of existing commercial and semi-integrated solutions to highlight where progress has been made and what gaps remain. Finally, we outline future opportunities in enzyme engineering, unified buffer formulation, and microfluidic design that may enable reliable one-pot systems and transform scRT-qPCR into a powerful and widely accessible tool for quantitative single-cell analysis in both research and clinical settings.