RT-qPCR for Astrocyte Gene Expression

RT-qPCR was carried out as previously described [52 (link)] and summed up in Table 2. The TRI-Reagent (Sigma-Aldrich) was added to primary astrocyte samples to extract total mRNA. An equal amount of mRNA (1 μg) from each experimental group was quantified by a D30 BioPhotometer spectrophotometer (Eppendorf AG, Hamburg, Germany) and reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). RT-PCR reactions (20 μL) were set mixing specific primers, cDNA (20 ng) with the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and run in 96-well plates using the CFX96 Touch thermocycler (Bio-Rad, Hercules, CA, USA). All samples were run concurrently in triplicate. A two-step thermal protocol was used for 40 cycles (10 s at 95 °C and then 30 s at 60 °C) preceded by a polymerase activation step (3 min at 95 °C). Gene-specific amplification was controlled by including a melting curve analysis of the amplification products at the end of the reaction. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as reference gene to normalize each target amplicon. Data were analyzed using the Pfaffl method, correcting the delta Ct values for each primer efficiency [53 (link)]. All primer sequences are listed in Table 2.