Astrocyte Gene Expression Analysis

After euthanasia by CO2 asphyxiation, brains were immediately extracted and kept ice-cold during dissection. Brains were sliced using rodent brain slicer matrix (Zivic Instruments, Pittsburg, PA). Cortex samples were collected from 2 mm central coronal sections of each brain. RNA isolation from cortex, cultured and acutely isolated astrocytes was performed as described before [50 (link)]. RNA samples were processed by the Institute for Genome Science at the University of Maryland School of Medicine using the nCounter custom-designed Nanostring gene panel (Nanostring Technologies, Seattle, WA), which consisted of genes that are expressed predominantly by astrocytes (www.brainrnaseq.org). Only samples with an RNA integrity number RIN > 7.2 were used for Nanostring analysis. All data passed quality control, with no imaging, binding, positive control, or CodeSet content normalization flags. The analysis of data was performed using nSolver Analysis Software 4.0. Ten house-keeping genes (Xpnpep1, Lars, Tbp, Mto1, Csnk2a2, CCdc127, Fam104a, Aars, Tada2b, Cnot10) were used for normalization of gene expression.