Superscript 2 first strand reverse transcriptase system

For qRT-PCR measurements, CD11b+ magnetic sorted microglia cells or embryonic neural progenitor cells were homogenized in QIAzol Lysis Reagent (#79306, QIAGEN) either immediately after isolation or after 10 days of in vitro culturing. Total RNA was isolated using Direct-zol RNA Miniprep Kits (#R2052, Zymo Research) following the manufacturer’s protocol. The RNA purity and concentration were assessed by NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies). The isolated RNA was then stored at −80°C. RNA was subjected to DNase I (#AM2224, Ambion) treatment in the presence of RNase H inhibitor (#AM2682, Ambion). Standardized quantities of RNA were reverse transcribed to cDNA using the SuperScript II First-strand Reverse Transcriptase system (#18064014, ThermoFisher Scientific) and random hexamers (#48190011, Invitrogen) supplemented with RNase H inhibitor (#AM2682, Ambion).

The otal RNA content was extracted using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. The RNA concentration and quality were determined by a spectrophotometer (NanoDrop ND1000; Promega Biosciences, Madison, WI, USA). The isolated RNA was treated with DNase and RNase inhibitor (Ambion, Austin, TX, USA). cDNA synthesis was done using random hexamers and the SuperScript II First-strand Reverse Transcriptase system (Thermo Fisher Scientific, Waltham, MA, USA).