Sed as a control. The Asensors were quantified by qPCR, and when the amount matched that of the Asensor-infected target cells, then the activity of miRNAs in cells paired with the target 10781694 sequences of the Asensor was revealed by Gluc expediently, accurately, and sensitively.Calculation of Transduction Coefficients and Relative Inhibiting FoldsAlthough equal amounts of each miRNA Asensor were loaded into each well of the 96-well plate, the Fluc activity reflecting the transduction efficiency remained variable among different Asensors due to fluctuations in the titre for each Asensor. To solve this problem, the transduction coefficient (TC) was used to calibrate the miRNA activity obtained by each Asensor. The TC calculation for each Asensor was performed as follows. Without consideration of the miRNA repression of Gluc activity, the relationship between Fluc activity (F) and Gluc activity (G) could be approximated by G 30:938F 1:3192 ??Then, the TC value, which is the ratio of G of AsensormiRNA (GmiRNA) to that of Asensorcontrol (Gcontrol) when no miRNA repression occurs, could be computed as follows: GmiRNA FmiRNA 1:3192 Gcontrol FcontrolTC??Cell CultureThree PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were obtained from the American Type Culture Collection and grown under the recommended conditions, supplemented with 10 fetal bovine serum (Invitrogen, Carlsbad, CA) and 100 mM each of penicillin and streptomycin (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 5 CO2 at 37uC.where Gcontrol and GmiRNA represent the Gluc activity of the Asensor control and Asensor miRNA, respectively, and Fcontrol and FmiRNA represent the Fluc activity of the Asensor control and Asensor miRNA, respectively. The activity of miRNA in a cell line, represented by the relative inhibiting fold (RIF), was calculated by the formula RIF Gcontrol FmiRNA 1:3192 Gcontrol | |TC ??GmiRNA Fcontrol GmiRNAmiRNA Monitoring in Pancreatic Cells Using AsensorRIF represents the level of report gene Autophagy expression regulated by miRNA compared with the control, which depends on the theory that “miRNAs inhibit target gene expression” [13].Statistical AnalysisGraphs were created with GraphPad Prism 6.0.1 software (San Diego, CA, USA). Differences in the RIF between miRNA and the control were tested for statistical significance by the independentsamples t test (SPSS version 17.0, Chicago, IL, USA). The level of significance was chosen as p,0.05.Results Assessment of Asensor ControlThe Asensor was constructed as described in the Materials and Methods section. The Gluc expression Asensor without miRNA binding sites was also constructed as a control. To ensure that all rAAVs could act as an miRNA sensor, we first evaluated the Asensor control, and Gluc secreted into the supernatant was assayed at 24, 48, 72 hours after infection. As expected, we could detect high Gluc and Fluc signals in all cells. In this report, the Epigenetics infection efficiency of the Asensors, cell growth diversity, and other system and accidental errors were calibrated by Fluc, and miRNA activity was expressed as Gluc/Fluc. Although the Asensor control did not reflect any miRNA activity, to maintain consistency, the y axis in Figure 1 was also expressed as Gluc/Fluc. As shown in Figure 1, the Gluc/Fluc of the supernatants of BxPC-3 cells was consistently high, measuring 1130.516350.93, 6633.1662931.6, and.Sed as a control. The Asensors were quantified by qPCR, and when the amount matched that of the Asensor-infected target cells, then the activity of miRNAs in cells paired with the target 10781694 sequences of the Asensor was revealed by Gluc expediently, accurately, and sensitively.Calculation of Transduction Coefficients and Relative Inhibiting FoldsAlthough equal amounts of each miRNA Asensor were loaded into each well of the 96-well plate, the Fluc activity reflecting the transduction efficiency remained variable among different Asensors due to fluctuations in the titre for each Asensor. To solve this problem, the transduction coefficient (TC) was used to calibrate the miRNA activity obtained by each Asensor. The TC calculation for each Asensor was performed as follows. Without consideration of the miRNA repression of Gluc activity, the relationship between Fluc activity (F) and Gluc activity (G) could be approximated by G 30:938F 1:3192 ??Then, the TC value, which is the ratio of G of AsensormiRNA (GmiRNA) to that of Asensorcontrol (Gcontrol) when no miRNA repression occurs, could be computed as follows: GmiRNA FmiRNA 1:3192 Gcontrol FcontrolTC??Cell CultureThree PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were obtained from the American Type Culture Collection and grown under the recommended conditions, supplemented with 10 fetal bovine serum (Invitrogen, Carlsbad, CA) and 100 mM each of penicillin and streptomycin (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 5 CO2 at 37uC.where Gcontrol and GmiRNA represent the Gluc activity of the Asensor control and Asensor miRNA, respectively, and Fcontrol and FmiRNA represent the Fluc activity of the Asensor control and Asensor miRNA, respectively. The activity of miRNA in a cell line, represented by the relative inhibiting fold (RIF), was calculated by the formula RIF Gcontrol FmiRNA 1:3192 Gcontrol | |TC ??GmiRNA Fcontrol GmiRNAmiRNA Monitoring in Pancreatic Cells Using AsensorRIF represents the level of report gene expression regulated by miRNA compared with the control, which depends on the theory that “miRNAs inhibit target gene expression” [13].Statistical AnalysisGraphs were created with GraphPad Prism 6.0.1 software (San Diego, CA, USA). Differences in the RIF between miRNA and the control were tested for statistical significance by the independentsamples t test (SPSS version 17.0, Chicago, IL, USA). The level of significance was chosen as p,0.05.Results Assessment of Asensor ControlThe Asensor was constructed as described in the Materials and Methods section. The Gluc expression Asensor without miRNA binding sites was also constructed as a control. To ensure that all rAAVs could act as an miRNA sensor, we first evaluated the Asensor control, and Gluc secreted into the supernatant was assayed at 24, 48, 72 hours after infection. As expected, we could detect high Gluc and Fluc signals in all cells. In this report, the infection efficiency of the Asensors, cell growth diversity, and other system and accidental errors were calibrated by Fluc, and miRNA activity was expressed as Gluc/Fluc. Although the Asensor control did not reflect any miRNA activity, to maintain consistency, the y axis in Figure 1 was also expressed as Gluc/Fluc. As shown in Figure 1, the Gluc/Fluc of the supernatants of BxPC-3 cells was consistently high, measuring 1130.516350.93, 6633.1662931.6, and.
