Detection 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.