mRNA Expression Analysis of GluN and GluA Receptors

For mRNA expression analysis on MBMECs, total RNA was extracted using a Quick RNA Micro Prep Kit (Zymo Research). cDNA was synthesized from 300 ng of total RNA using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific). All experiments were performed according to the manufacturers’ instructions and as described before [16 (link)]. For quantitative real-time PCR (qPCR) 4 µl cDNA were used together with Maxima Probe Rox qPCR mix supplemented with mouse GluN1 (Mm00433790_m1), mouse GluN2A (Mm00433802_m1), mouse GluN2B (Mm00433820_m1), mouse GluA1 (Mm00433753_m1), mouse GluA2 (Mm00433753_m1), mouse GluA3 (Mm00497506_m1), mouse GluA4 (Mm00444755_m1) and 18S rRNA (Hs99999901_s1) as endogenous control for TaqMan gene expression assays. For qPCR with QuantiTect Primers 4 µl of cDNA were used together with a SYBR green master mix for GluN2C (QT00127015), GluN2D (QT00154378), GluN3A (QT00290843) and GluN3B (QT00173684) expression assays. 18S rRNA was used as endogenous control. All qPCRs were performed using the StepOnePlus System for 40 cycles (Applied Biosystems). Data were calculated using the change in cycle threshold (ΔCT) compared to the 18sRNA [17 (link)].