Fig. 5: Using BFQs and PBCV-1 DNA ligase for multivariate and quantitative detection of IVT-mRNA production.

a Principle of multiplex RT-IVT method. Figure was created with Biorender (www.bioender.com). b The target-signal standard curves for BFQ1, BFQ2 and BFQ3. The FRET signal represents the fluorescence difference between each group and the 0 nM cDNA group (control). c‒e The raw, relative quantitative corrected signal and absolute quantitative corrected signal results for the three DNA templates with their transcriptional activities. 200 nM T7 RNA polymerase; 30 nM DNA template 1, DNA 2 and DNA 3; 500 µM NTP mixture; 500 nM BFQ1, BFQ2 and BFQ3; and 50 nM PBCV-1 DNA ligase were mixed in standard reaction buffer and immediately and simultaneously tested in a RT-PCR thermocycler at 37 °C using its FAM/VIC/ROX scan channels. The orange square and red circles in Fig. 5e represent the transcriptional activity results for DNA template 1 tested by the spectrophotometric and the RT-PCR methods. Between the groups, the relative quantitative detection results for the RT-PCR method corresponded to the right, red coordinate axis. The values represent the mean ± standard deviation of three repeats.