Figure 3From: 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 qPCR. In vitro transcription was performed on the pEGFP-N1 plasmid (A, C) or linearized CMV-EGFP template (B, C, D). The RNA was purified, reverse transcribed and detected by qPCR. A. qPCR amplification curves of the primer-extension products from A as detected by a sequence-specific LNA probe. The C(T) value of the α-amanitin-inhibited probe (+αA 100 μM) of 22.3 was significantly lower than that of the probe generated without amanitin (−αA, c(T) = 17.6) indicating significantly lower RNA amounts. B. qPCR amplification of RNA/cDNA after in vitro transcription from the linear template CMV-EGFP in the presence of 5-100 μM α-amanitin showed no clear α-amanitin-concentration dependency. -αA – positive transcription (without α-amanitin, C(T) = 16.1); +αA – α-amanitin-inhibited transcription, C(T) values 27.9, 37.0, 28.1 and 24.5 for 100, 50, 20 and 2 μM, respectively; −NTP/-αA – negative control transcription; DNase - DNase digested DNA control. C. Amplification curves of RNA products from three different experiments (Exp1-Exp3). Positive transcription reactions performed at different occasions resulted in very similar C(T) values of 17.6, 18.0 and 16.1 for Exp1, 2 and 3, respectively. No amplification occurred for the negative controls. Note that the inhibitory effect of α-amanitin was dramatically increased when the linearized template CMV-EGFP was used for transcription (Exp3, C(T) = 27.9), when compared to Exp1 and Exp2 (C(T) values 22.3 and 21.7, respectively), were the supercoiled plasmid pEGP-N1 was used as a template. αA – transcription without α-amanitin; +αA - transcription in the presence of α-amanitin; −NTP/-αA – negative control transcription. D. Semi-quantitative estimation of the relative inhibitory activity of α-amanitin and several semisynthetic and synthetic analogs. 6–O-Met-γ-Ama = 6′-O-methyl-γ-amanitin; Amanitin = α-amanitin. Two different charges of the analog HDP30.0470 were tested.Back to article page