ArticleCommunications biology2023
RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.
Article in Communications biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed.
- DNA Flap-Mediated Control of Transcription for Programmable RNA Synthesis.Angewandte Chemie (International ed. in English) · 2026Article
- Nucleolar migration regulates meiotic sex chromosome inactivation via phase separation during mammalian spermatogenesis.Nature communications · 2026Article
- Macromolecular crowding alters transcription: real-time measurements with SYBR Green II.Research square · 2026Article
- In-process analytics for IVT mRNA manufacturing: from process understanding to advanced process control.Frontiers in bioengineering and biotechnology · 2026Review
- A NanoLock-enabled, Craspase-based strategy for highly sensitive RNA detection.Nucleic acids research · 2025Article
- Real-time monitoring strategies for optimization ofFrontiers in molecular biosciences · 2023Review
Corrections and comments
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.
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Registered trials
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