Transcription was carried out as described previously (30 (link)). When a T7 transcription was followed by a T3 transcription, the T7 transcription was stopped by adding a final concentration of 2 μM T7 inhibitor (5′-GAAATTAATACGACTCACTATA-3′) (31 (link)). Then, 0.02 mM Fluorescein-12-UTP (Roche) and 2 U/μl T3 RNA polymerase (Thermo Scientific) were added. The samples were incubated at 37°C for 60 min, followed by a treatment with 0.04 U/μl DNase I (Thermo Scientific) at 37°C for 15 min and an extraction with phenol/chloroform. RNA products were denatured in 80% formamide and resolved on an 8% denaturing polyacrylamide gel.
Protocol detail
Fluorescent RNA Transcription Assay
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Corresponding Organization : Sun Yat-sen University
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Variable analysis
independent variables
- Presence or absence of T7 inhibitor (5′-GAAATTAATACGACTCACTATA-3′)
- Concentration of Fluorescein-12-UTP (0.02 mM)
- Concentration of T3 RNA polymerase (2 U/μl)
- RNA products
- Transcription protocol (as described previously)
- Incubation temperature (37°C)
- Incubation time (60 min for T3 transcription, 15 min for DNase I treatment)
- Gel electrophoresis conditions (8% denaturing polyacrylamide gel)
- Positive control: T7 transcription followed by T3 transcription without T7 inhibitor
- Negative control: T7 transcription followed by T3 transcription with T7 inhibitor
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