A novel in vitro transcription assay using DNA template depletion, primer extension and qPCR.

(A) Schematic of the approach used for the DNA template depletion. (B) A standard curve plotted with average Ct value against—log of DNA template quantity (ng). The standard curve was generated by using DNA constructs containing the luciferase gene (pGL3-E4) and qPCR performed with reporter gene primers(LF/LR), the standard curve was used to determine the quantity of DNA template after depletion. (C) A scheme for detecting in vitro transcription products using specially designed primers; ① represents the transcription products after the DNA template depletion, which includes the remaining DNA template (circle) and RNA transcript (wave line), ② represents primer extension using a GFP oligonucleotide-tagged luciferase gene reverse primer, in which the GFP oligonucleotide (grey line) linked with the luciferase oligonucleotide ((-))is complementary to luciferase gene RNA transcript (the black straight line and (+)). ③ represents qPCR using cDNA template and the luciferase gene forward primer (black arrow) and GFP gene reverse primer (grey arrow); (-) represents the cDNA template derived from the primer extension, (+) represents the DNA template from the first amplification of the cDNA. LF/LR: Luciferase gene primer pair. (D) Transcription analyses of the E4 promoter under conditions with (E4a) or without the activator GAL4-AH (E4b). (E) Transcription analysis of the ML promoter with (MLa) or without the activator GAL4-AH (MLb). Each bar represents the mean of at least three independent experiments with standard deviation, the symbol “* *” represents P≤0.01, the p values were obtained by performing t test.