Massarray compact system

Genomic DNA was isolated from ethylenediaminetetraacetic acid (EDTA) blood samples using the QIAmp^® DNA Blood Mini Kit System (Qiagen, Hilden, Germany). Candidate genetic variants of SDF1 were selected on the basis of previous literature reporting on either clinical importance or functional importance on protein expression profile. The criteria for selection of variants in these genes were a representative allele frequency in Caucasians and clear evidence for functional consequences based on in vitro/in vivo data.[22 (link),23 (link),24 (link),25 (link),26 (link)] Thus, the following polymorphisms of SDF1 were analysed: rs1065297, rs2839693, rs1801157, rs266087, rs266085 and rs266089. Genotyping for SDF1 variants was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the MassARRAY^® Compact system (Sequenom, CA, USA) as previously described.[27 (link)] Details of primers and assays are available upon request. Approximately 10% of samples within each assay were retyped as a quality control. Study personal assessing outcome was blinded to the case status of the study participants during the entire genotyping process. Minor allele frequencies of SDF1 variants in the study cohort are provided in S1 Table. Linkage disequilibrium (LD) map is shown in S1 Fig.

10 nL of the resultant cleavage reactions was spotted onto silicon matrix‐preloaded chips (Spectro‐CHIP; Sequenom) using a MassARRAY nanodispenser (Sequenom) and analyzed using the MassARRAY Compact System matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometer (MALDI‐TOF) (Sequenom). The spectra's methylation ratios were calculated using epityper software v1.0 (Sequenom). The method yields quantitative results for each of the sequence‐defined analytic units referred as CpG units, which may contain either one individual CpG site or an aggregate of downstream CpG sites. Triplicate independent analyses from sodium bisulfite‐treated DNA sample were undertaken.
The effectiveness of the entire experimental procedure was assayed by analyzing as control CpGenome Universal Unmethylated DNA (Chemicon) and CpGenome Universal Methylated DNA (Chemicon, Millipore, Germany) in serial mixtures of methylated and unmethylated products, with 10% methylation increments.
Data quality control and filtering were carried out by the removal of the CpG dinucleotides whose the measurement success rate was <80%. Poor‐quality and nonvaluable data for the quantitative methylation of each CpG unit measured by MALDI‐TOF‐MS were excluded.

A 3 kb DNA sequence from −2000 to +1000 of the SSTR-2 (GeneBank NC_005109.4, 102136283–102143449) transcriptional start site was subjected to an analysis of CpG islands using MethPrimer (http://www.urogene.org/methprimer) with the following parameters: island size > 200 bp, GC Percent > 50.0, Obs/Exp > 0.6. Genomic DNA was extracted using the Blood and Tissue DNA purification kit (Qiagen) and subjected to bisulfite modification using EpiTect Bisulfite Kits (Qiagen) according to the manufacturer’s protocol. Primers were designed using MethPrimer (http://www.urogene.org/methprimer/) and Primer Premier 5.0 (Premier, Canada), as shown in Table 1. Sequences of interest were then amplified using HotStar Taq DNA Polymerase (Qiagen). The Sequenom MassARRAY was followed by in vitro RNA transcription and base-specific cleavage (MassCCLEAVE Kit, San Diego, USA, SEQUENOM), according to the manufacturer’s protocol. Mass spectra were analyzed using the MassARRAY Compact System (SEQUENOM). Methylation ratios were then generated by the EpiTYPER software (SEQUENOM). The Sequenom methylation analysis was performed at CapitalBio, Beijing, China.