7900 abi real time instrument

To quantify cell type-specific gene expression, we performed standard qRT-PCR. Briefly, nuclear RNA was reverse transcribed into cDNA by Superscript III reverse transcriptase (Life Technologies) with random primers. RNA expression levels were determined by qRT-PCR using gene-specific primers and a 7900 ABI real-time instrument (Applied Biosystems). SYBR Green SuperMix was used for all cell-specific gene targets and a TaqMan gene expression assay kit for human actin beta (ACTB) was used as an internal control (ThermoFisher, Assay # Hs99999903_m1). All samples were performed in duplicate and cycle numbers were averaged. Primer sequences (Supplemental Table 7b) were obtained from previously published work including allograft inflammatory factor 1 (AIF1) (Lorente-Cebrian et al., 2013 (link)), 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNP) (Croitoru-Lamoury et al., 2011 (link)), and glial fibrillary acidic protein (GFAP) (Yang et al., 2015 (link)). Microtubule-associated protein 2 (MAP2) primers were designed in-house using Primer 3 online software (http://bioinfo.ut.ee/primer3). Relative gene expression levels were calculated using the ΔΔCt method with ACTB as a reference gene.

We performed qRT-PCR as previously described [13 (link)]. Briefly, total RNA (2 μg) was reverse transcribed into cDNA by Superscript III reverse transcriptase (Life Technologies, Carlsbad, CA) with random primers. RNA expression levels were determined by real-time qPCR using TaqMan gene expression assays, TaqMan Universal PCR master mix, and a 7900 ABI real-time instrument (Applied Biosystems). TaqMan assays were used to quantify mRNA levels of human APOE and beta-actin (ACTB). Twenty-four independent frontal lobe samples were run in triplicate with ACTB as an endogenous control. Relative expression levels were determined by normalizing all samples to the lowest APOE expressing sample as determined by ΔCt (set at 1.0) using the ΔΔCt method.