The intronic SNPs rs3796902, rs1947187, and rs2595110 in PITX2 were screened from the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/ ) based on their MAF (minor allele frequency) (≥ 10% in the global population).
The genomic DNA of each included patient was used for the genotyping analysis. The DNA was isolated from epithelial cells collected with cytobrushes using extraction solution (Tris-HCl 10 mmol/L, pH 7.8; EDTA 5 mmol/L; SDS 0.5%, 1 mL) and proteinase K (100 ng/mL) and ammonium acetate to remove non-digested proteins. The DNA was precipitated with isopropanol and resuspended, and later quantified by spectrophotometry (Nanodrop 1000; Thermo Scientific, Wilmington, DE, USA) [23 (link)]. All 3 SNPs were blindly genotyped using real-time Polymerase chain reaction (real time-PCR) in the Mastercycler® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany). The TaqMan technology, which uses extremely sensitive allele-specific probes (VIC™ and FAM™ dyes were used for the alleles), was used in this study. One negative control template (omitting the DNA) was used in each reaction plate. Additionally, 10% of the samples were randomly selected for repeated analysis (presented 100% concordance). DNA samples that failed to be genotyped were considered missing data and was excluded from the statistical analysis.
The genomic DNA of each included patient was used for the genotyping analysis. The DNA was isolated from epithelial cells collected with cytobrushes using extraction solution (Tris-HCl 10 mmol/L, pH 7.8; EDTA 5 mmol/L; SDS 0.5%, 1 mL) and proteinase K (100 ng/mL) and ammonium acetate to remove non-digested proteins. The DNA was precipitated with isopropanol and resuspended, and later quantified by spectrophotometry (Nanodrop 1000; Thermo Scientific, Wilmington, DE, USA) [23 (link)]. All 3 SNPs were blindly genotyped using real-time Polymerase chain reaction (real time-PCR) in the Mastercycler® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany). The TaqMan technology, which uses extremely sensitive allele-specific probes (VIC™ and FAM™ dyes were used for the alleles), was used in this study. One negative control template (omitting the DNA) was used in each reaction plate. Additionally, 10% of the samples were randomly selected for repeated analysis (presented 100% concordance). DNA samples that failed to be genotyped were considered missing data and was excluded from the statistical analysis.
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