Real-time quantitative PCR analysis of human astrocytes exposed to pressure-driven flow and/or Direct Current Stimulation in vitro

The objective of this study was to investigate the effect of Direct Current Stimulation (DCS) on the gene expression of human astrocytes (HA). DCS at 0.1 or 1mA was applied to monolayers of HA for 10 minutes. Expression of a set of neuroactive genes was assessed immediately of 1 hour after DCS using RT-qPCR. Because DCS can produce electroosmotic flow and fluid shear stress known to influence cell function, we compared three interventions: pressure-driven flow across the monolayer alone (conP), pressure-driven flow plus DCS (DCS P), and DCS alone with flow blocked (DCS S). HA were obtained from Cell Applications. DCS was applied with (DCS P) or without (DCS S) a pressure differential that induced fluid flow across the cell monolayer. Monolayers exposed only to convective flow (conP) were also examined. A static control (conS) kept in an incubator for the duration of the experiment was used as the calibrator sample for gene expression analysis. Total RNA was collected either immediately after DCS, or 1 hour after DCS using the RNeasy Mini kit from Qiagen, and reversed transcribed using the High-capacity cDNA Reverse Transcription kit from Thermo Scientific. Real time RT-qPCR was performed on the 7300 Real Time PCR System from Applied Biosystems. Samples were run in duplicates.