Figure 10.

Improved giga seal during harvesting of cytosol using β-escin as pore-forming agent. The grey dots in each chart representing individual Cq values of the harvested cytosol from each cell with black lines representing mean ± SD. To evaluate and visualize the impact of the pore-forming agents amphotericin B and β-escin on preserving the giga seal during harvesting, we used EF1α as the target gene. The assay had an upper Cq detection limit of 37, and only samples where stable giga seal allowed cytosol to be collected for more than 10 s were analyzed. First chart column. Use of amphotericin B regularly lead to disruption of the giga seal soon after breaking the patch by suction, thus allowing harvesting for only a short period of time. The mean Cq value of EF1α using amphotericin B was 34.94 ± 1.9 (n = 36) with 11 of the samples reaching the upper Cq detection limit. Second chart column. Changing to β-escin significantly reduced the mean Cq to 32.04 ± 0.92 (n = 13) (p < 0.0001 ***), allowing most of the available RNA to be harvested. The mean difference in Cq was 2.91 ± 0.41 (mean ± SEM). The variance was also significantly different (F-test with P = 0.0051).