Investigations of convergent and divergent synthesis of Dome11, GFP, Sec23A, and 120 bp dsRNA in vitro using run-off transcription. All Dome11, GFP, Sec23A, and 120 bp plasmids constructs were investigated. (A) Agarose gel electrophoresis analysis of RNA extractions in the presence of RNase T1 and DNase. The corresponding Dome11 (400 bp), GFP (263 bp), Sec23A (1504 bp), and 120 bp dsRNA and potential multimers are highlighted. (B) Quantification of dsRNA yield. RNA samples were generated from 20 µL IVT reactions, followed by the addition of RNase T1 and DNase prior to purification. RNA samples were analyzed using UV spectrophotometry to determine RNA concentration using a mass concentration/A260 unit (46.52 μg/mL). Significance was calculated using a one-way ANOVA test with multiple comparisons for Dome11 RNA samples. Divergent-produced Dome11 (pD11_D_1kb) demonstrates the highest absolute yield of 67.44 μg. Significance was calculated using unpaired t-tests, for GFP, Sec23A, and 120 bp RNA samples. Divergent-produced, GFP (pGFP_D), Sec23A (pSec23A_D), and 120 bp (p120_D) demonstrate the highest absolute yields of, 37.61, 42.13, and 12.63 μg, respectively. Data are shown as bar charts of triplicate technical replicates; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.
