Figure 2From: A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activityDetection of newly synthesized RNA by primer extension and PCR. In vitro transcription was performed on the pEGFP-N1 plasmid (A) or linearized CMV-EGFP template (B). The RNA was purified, reverse transcribed and detected by PCR. A. Gel analysis of the PCR products demonstrating α-amanitin-sensitive RNA synthesis. 1- DNA ladder; 2- no α-amanitin; 3- 100 μM α-amanitin; 4- negative transcription control (without NTPs); 5- positive PCR control; 6- DNase digested DNA control; 7- water PCR control. B. The visualized PCR product amount inversely correlated with the α-amanitin concentration. 1- DNA ladder; 2- no α-amanitin; 3-5- 400, 100 and 20 μM α-amanitin, respectively; 6-8- 400, 100 and 20 μM synthetic amanitin analog 30.0346, respectively; 9- negative transcription control (without NTPs); 10- positive PCR control; 11-water PCR control.Back to article page