Quickgene auto240l

Genomic DNA was extracted from peripheral blood using an automated DNA extraction system (QuickGene-Auto240L, Kurabo, Japan). Repeat expansion in NOTCH2NLC was examined by repeat-primed PCR, followed by electrophoresis on a 3500xl Genetic analyzer (ThermoFisher Scientific, Waltham, MA, USA), as described previously [6 (link)]. Briefly, PCR was performed in a total volume of 10 μL of reaction solution containing 0.25U PrimeSTAR GXL DNA Polymerase; 1 × PrimeSTAR GXL Buffer; 200 µM of dATP, dTTP, dCTP (Takara Bio, Shiga, Japan), and 7-Deaza-2′-deoxy-guanosine-5′- triphosphate (Sigma-Aldrich, St. Louis, MO, USA); 5% dimethyl sulfoxide (Sigma-Aldrich); 1 M betaine (Sigma-Aldrich); 0.3 µM of each primer mix; and 100 ng of genomic DNA. The presence of a sawtooth pattern in the electropherogram was regarded as an abnormal repeat expansion. NOTCH2NLC repeat size was further determined by fluorescence amplicon-length PCR as described previously [6 (link)]. GeneMapper software (ThermoFisher Scientific) was used to determine GGC repeat length.

The height and weight of all participants were measured, and the body mass index (BMI) was calculated. Blood was collected under fasting conditions in the morning, and the following blood variables were measured in all study participants: albumin (Alb), fasting blood glucose (FBG), and creatinine. In addition, the following blood variables were measured in all MCI and CN participants: white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb), hematocrit (Ht), platelet count, total protein (TP), prealbumin (TTR), C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), urea nitrogen, uric acid, calcium, iron, folate, total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride, insulin, and glycated hemoglobin (HbA1c).
Genomic DNA samples were extracted from peripheral blood leukocytes of all MCI participants using an automated DNA isolation system (QuickGene-Auto240L, Kurabo, Osaka, Japan). APOE genotypes (rs429358 and rs7412) were determined using TaqMan^® PCR Assays (Applied Biosystems, Foster City, CA, USA). According to previous studies [21 (link),22 (link)], we defined participants with ε4 allele as the APOE-positive group and those without ε4 allele as the APOE-negative group.