7900ht analyzer

RNA was extracted from spleens using Purelink RNA mini kit (Ambion). Gene expression was assessed using FAM-conjugated TaqMan assays as listed in the Supplementary Methods (online only, available at www.exphem.org). Template RNA was added in duplex reactions in triplicate using B2m VIC primer limited probe (Mm00437762_m1) as the endogenous control using the EXPRESS One-Step Superscript qRT-PCR Kit (Thermo Scientific) and an Applied Biosystems 7900HT analyzer. Relative gene expression between endogenous control and target gene was analyzed using the ΔΔCT method [10] (link) with RQ manager (Life Technologies) applying automatic thresholds.

In the European memory centers, APOE genotype was determined using standard polymerase chain reaction and restriction enzyme digestion according to established standard protocols [21 (link)].
In the ADNI study, APOE genotypes were determined by using DNA extracted by Cogenics from a 3-mL aliquot of EDTA blood. Polymerase chain reaction amplification was followed by HhaI restriction enzyme digestion, resolution on 4% Metaphor Gel, and visualization by ethidium bromide staining [22 (link)].
In Whitehall II and the Three-City Study, two TaqMan assays (Rs429358 and Rs7412, Assay-On-Demand, Applied Biosystems) were used and run on a 7900HT analyzer (Applied Biosystems), and APOE genotypes were indicated by the Sequence Detection Software version 2.0 (Applied Biosystems) [23 (link),24 (link)].

DNA was extracted from whole blood samples, drawn at the 1997–1999 clinical examination. Two TaqMan assays (Rs429358 and Rs7412, Assay-On-Demand, Applied Biosystems) were used and run on a 7900HT analyzer (Applied Biosystems) and genotypes indicated by the Sequence Detection Software version 2.0 (Applied Biosystems). Genotyping was repeated for 511 participants and error rates were found to be lower than 0.15% [28 (link)].