Quantitative Gene Expression Analysis of Neural Cells

Excised tissues sliced into ∼2-mm² pieces and preserved in RNA Later (QIAGEN) were mechanically dissociated in 500 μL Qiazol lysis reagent (QIAGEN) using a homogenizer (OMNI International), and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN). Alternatively, hippocampus and SVZ regions were dissected and dissociated using the adult brain dissociation kit from Miltenyi Biotec and the gentleMACS Octo Dissociator with heaters. Cells were then sorted on a FACSDiva 8.0.1 (BD Biosciences) directly into lysis solution (QIAzol) using the following antibodies as reported (Daynac et al., 2013 (link)): fluorescein isothiocyanate mouse LEX/CD15 (BD Biosciences, cat. no. 560127, clone MC480), PE rat CD24 (BD Biosciences, cat. no. 553262, clone M1/69), Alexa 647-conjugated EGF (Molecular Probes E35351). One μg or less of RNA was then reverse-transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative differences in gene expression were determined by real-time qPCR using SYBR Green PCR Master Mix (Bio-Rad) and a spectrofluorometric thermal cycler (Mx3000P from Stratagene). Values are presented as the ratio of target mRNA to 18S rRNA obtained using the relative standard curve method of calculation. Primers sequences were described previously (Le et al., 2010 (link)).