Zymo spin iiicg columns

To isolate RNA from mouse femora, bones were pestled in liquid nitrogen, homogenized using a planetary ball mill, vortexed in ribozol (VWR) for 45 min at room temperature, and centrifuged. The supernatant was extracted with chloroform. After adding an equal volume of ethanol, RNA was purified using Zymo-Spin IIICG columns (Zymo Research). RNA purification from primary microglia was performed using a Direct-zol RNA Microprep Kit (Zymo Research). BT-474 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). cDNA was synthesized by reverse transcription. Quantitative PCR was performed using the Light-Cycler 480 Probes Master system (Roche). The following primers were used: for human plxnb1: forward 5′-GACCGAGGTGGCCTACATCGAG-3′ and reverse 5′-ACCTTCAGAAGTGTGCTCTGGGTCATG-3′ (“primer pair #1”), and forward 5′-CGGGACCGCTGCAAGAAGGAATTC-3′ and reverse 5′-TCCACAGTGGGCCGTCTGCTC-3′ (“primer pair #2”); for mouse plxnb1: forward 5′-GTGTGCTGGAGCTAGGGAGTCGG-3′ and reverse 5′-CATGCAGCCCATCGGCACTG-3′ (“primer pair #1”), and forward 5′-GCCCGAGGAGCAGCGAGTG-3′ and reverse 5′-TCCTCCCCGCTGGCTCC-3′ (“primer pair #2”).