Faststart taq dna polymerase system

PCR and sequencing primers (Supplementary Table S9) were designed using the PyroMark Assay Design 2.0 software (Qiagen). PCR reactions were performed in a total volume of 25 µl using the FastStart Taq DNA Polymerase system (Roche Diagnostics, Mannheim, Germany). The 25-μl reaction consisted of 2.5 μl 10x PCR buffer, 20 mM MgCl₂, 0.5 μl dNTP (10 mM) mix, 10 pmol of forward and reverse primer, 1 IU of FastStart Polymerase (Roche Diagnostics), 1 μl (approximately 100 ng) bisulfite converted template DNA, and 18.8 μl PCR-grade water. To reduce technical noise (batch effects), bisulfite conversion and PCR (of matched DS and control samples) were performed in 96-well microtiter plates. Pyrosequencing was performed on a PyroMark Q96 MD system with PyroMark Gold Q96 CDT reagents (Qiagen). Methylation values were quantified using the Pyro Q-CpG software. The average methylation difference between technical replicates was approximately one percentage point. To obtain a common value for each gene the β values of CpG sites have been averaged over the gene for each tissue separately. To account for a potential effect of gestational age a linear model was subsequently fitted to each gene and tissue separately including chromosomal status and gestational age as covariates. All P values have been corrected for multiple testing using the method of Bonferroni-Holm.⁷³ (link)