Mastercycler ep realplex s thermocycler

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 DNA of hPDL cell samples was isolated using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Munich, Germany). DNA extraction was performed according to the manufacturer’s instructions. For purity and quantification of the DNA, optical density was measured at 260 nm and 230 nm (NanoPhotometer N60, Implen, Munich, Germany). The OD_260nm/280nm ratio > 1.8 indicated protein-free DNA.
A total of five SNPs in the VDR gene, which is located in 12q13.11, were selected based on their minor allele frequency (>10%) and their previously reported association. The SNPs’ characteristics are presented in Table 1. Genotyping was performed by real-time PCR using the TaqMan assay in the Mastercycler^® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany). The SNPs were blindly genotyped using TaqMan assay. PCR reactions were performed in a total final volume of 5 μL (2.5 μL Taqman genotyping master mix, 0.125 μL SNP assay; Applied Biosystems, Foster City, CA, and 4 ng DNA/reaction in water). Thermal cycling was performed by starting with a hold cycle of 95 °C for 10 min, followed by 40 amplification cycles of 92 °C for 15 s and 60 °C for 1 min.

The used oligonucleotides were designed based on the gene sequences achieved from the Nucleotide database NCBI (GeneBank, National Centre for Biotechnology Information) and validated for absence of secondary structures, self and cross dimers as well as primer efficiency and specificity, as already described [8 (link)] (Table 1). Eurofins MWG Operon LLC (Huntsville; High Purity Salt Free Purification HPSF^®) was assigned for primer synthesis and purification.
For each RT-qPCR reaction we used 7.5 μl SYBR^®Green JumpStart^™ Taq ReadyMix^™ (Sigma-Aldrich), 10 pmol/μl of the respective forward and reverse primer and 1.5 μl of the diluted cDNA (1:10). RNase-free H₂O (Carl-Roth) was added to a total volume of 15 μl. All cDNA samples were tested as three replicates per housekeeping gene and on the same 96 well PCR plate per biological replicate in 45 cycles (95°C for 5 min, per cycle 95°C for 10 s, 60°C for 8 s, 72°C for 8 s) to reduce possible inter-run variations on relative housekeeping gene stability assessment. Non-template controls and reverse transcription controls were additionally performed. For qPCR a Mastercycler^® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany) was used in conjunction with 96-well PCR plates (Biozym Scientific) covered with BZO Seal Filmcover sheets (Biozym Scientific) [8 (link),30 (link),31 (link)].