Pyromark q24 advanced cpg reagents kit

Prior pyrosequencing marker-specific PCRs were performed either using the HotStarTaq Kit (Qiagen, Hilden/Germany) or the PyroMark PCR Kit (Qiagen, Hilden/Germany) under manufacturer’s conditions. Primer sequences were taken from the original papers [29 (link)–31 (link)]. Hereafter, 10–20 µl of biotinylated PCR product was immobilized to 1 μl Streptavidin Sepharose™HP beads (GE Healthcare, Chicago, Illinois/USA). Sequencing primers were designed as described previously [29 (link)–31 (link)]. Pyrosequencing was performed using the PyroMark Q24 Advanced CpG Reagents Kit (Qiagen, Hilden/Germany) and the PyroMark Q24 Advanced System (Qiagen, Hilden/Germany).

To validate WGBS cytosine methylation results, we performed pyro-sequencing for several methylation sites in the D-Loop and ND4L regions of the mitochondrial genome. Individual blastocysts (n = 6) produced in vitro as described previously were treated with sodium bisulphite directly using the EZ DNA Methylation-Direct kit. PCR was performed for 2 regions on mtDNA, namely the D-loop and ND4L, in 25 µL using the PyroMark PCR kit (Qiagen) in 1X buffer, 3 mM MgCl₂ and 5 µL bisulphite-modified DNA (corresponding to 0.1 blastocyst). Primers information are presented in Suppl Table 1). The cycling conditions were as follows: 95 °C for 15 min followed by 45 cycles of 30 sec at 95 °C; 30 sec at 52 °C; 30 sec at 72 °C; and a final elongation step of 10 min at 72 °C. PCR products were verified by electrophoresis on 2% w/v agarose gel and then attached to streptavidin-coated Sepharose beads (GE Healthcare) in the presence of binding buffer (Qiagen). The pyro-sequencing reactions were performed on a PyroMark Q24 Advanced system using the PyroMark Q24 advanced CPG reagents kit (Qiagen) in accordance with the manufacturer’s recommendations. The pyrosequencing assay design was done using the Pyromark assay design 2.0 (Qiagen).