Pyromark pcr kit

The PCR primers 5′-gtaatttagtggtgttgttgaat-3′, 5′-biotin-cctaacccccaaccaacttcttactac-3′ and the sequencing primer 5′-gggttgagttaagtgtgtttggtaga-3′ for the region chr10:1405336-1405409 (hg19) were designed with Qiagen PyroMark AssayDesign SW 2.0. The samples were prepared with Qiagen EpiTect Fast Bisulfite Conversion and Qiagen PyroMark PCR kits and sequenced with Qiagen PyroMark Q24 Advanced system. The amplicons were analyzed with PyroMark Q24 Advanced software version 3.0.0. The statistical analysis was carried in R v3.4.3 [46 ] using packages lme4 v1.1-15 [20 (link)], car v2.1-6 [57 ], survival 2.42-4 [58 ], coxme 2.2-10 [59 ], and ggplot2 v2.2.1 [60 ]. Association of AD with CpG methylation was examined with lme4 package, with modeling methylation level as a function of disease status. Influence of covariates and their interaction with AD in the model was examined, including gender, age, zygosity, APOE genotype, and smoking (never, ever) as fixed effects and twin pair nested with genomic position and country of origin as random effects as specified in the results. The model with the lowest AIC was chosen. The Cox mixed effects model with coxme package was utilized to examine CpG methylation level as a prognostic marker of disease outcome. In this model, only age and gender were included as fixed effects and twin pair information nested with genomic position as a random effect.

Quantification of DNA methylation levels by pyrosequencing was performed. Briefly, genomic DNA (500 ng each) was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen). To evaluate the methylation status of the APOE’s DMR 1, we designed pyrosequencing assays to cover 27 CpG sites (CpG 11–37). PCR was performed on approximately 200 ng of bisulfite-converted DNA using PyroMark PCR kits (Qiagen) on a GeneAmp PCR System 9700 (Applied Biosystems, Grand Island, NY). Pyrosequencing was carried out on a PyroMark Q24 system (Qiagen) and data was analyzed using PyroMark Q24 software, version 2.0.6 (Qiagen). Bisulfite treatment controls were integrated as a quality control measure. Detailed procedures have been described in our prior published works [47 (link)]. Biological samples untreated and two independently treated with 5-aza-dC were used for bisulfite pyrosequencing.

Select differentially methylated genes as determined by NimbleGen methylation array were validated by pyrosequencing. Genomic DNA was treated with sodium bisulfite using EpiTech Bisulfite Kit (Qiagen). Pyrosequencing PCR and sequencing primers for select differentially methylated genes were designed using PyroMark Assay Design version 2.0.1 software (Qiagen) (Table S1). PCR amplification of bisulfite-converted genomic DNA was done using PyroMark PCR kit (Qiagen) under conditions as specified by the manufacturer. PCR products were submitted to Stanford University Protein and Nucleic Acid Facility (Palo Alto, CA) for pyrosequencing. Quantitative methylation analyses were done using PyroMark Q23 version 2.0.6 software (Qiagen).