biostudies-literatureZhang FNatural Science Foundation of Shandong Province (Shandong Provincial Natural Science Foundation)Natural Science Foundation of Shandong ProvinceNational Natural Science Foundation of ChinaChina Postdoctoral Science FoundationNational Natural Science Foundation of China (National Science Foundation of China)453https://www.ebi.ac.uk/biostudies/studies/S-EPMC10124930biostudies-literatureUnknown6(1)For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexedCommunications biologyRT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.PMC10124930ZR2021022306752021M70199631970043Zhang FZhang KDong HChen MHu WGu LWang XWang HWang YDu NfalseRT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed2023-01-01T00:00:00Z2023 Apr2025-04-22T18:04:59.368Z2025-04-22T18:04:59.368ZS-EPMC101249303709529210.1038/s42003-023-04830-1