Pyromark gold reagents kit

Genomic DNA samples were isolated from primary human LAM cell lines and normal human lung fibroblast cells. For DNA isolation, cells were lysed in 0.5% SDS-containing TE buffer (pH 8.0) followed by Proteinase K treatment at 55°C overnight. Genomic DNA was then isolated using standard phenol-chloroform method and re-suspended in TE buffer. One microgram of each DNA sample was bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen Sciences, Inc., Germantown, MD) following manufacturer's protocol. Pyrosequencing was performed to analyze the methylation profiles at the H19/IGF2 imprinting control region (ICR) as described previously [21 (link)]. 50ng of bisulfite-treated DNA was used for PCR. The PyroMark PCR kit (Qiagen Sciences, Inc., Germantown, MD) was used in a 25μL reaction according to the manufacturer's protocol. PCR conditions were: 95°C for 15 minutes followed by 45 cycles of 95°C for 30 seconds, 55° for 30 seconds and 72°C for 30 seconds, and 5 minutes of extension at 72°C. 10μL of the biotinylated PCR product was used for pyrosequencing. Pyrosequencing was done using the PyroMark Q96MD (Qiagen Sciences, Inc., Germantown, MD) system following the manufacturer's protocol and the PyroMark Gold reagents kit (Qiagen Sciences, Inc., Germantown, MD). Methylation was analyzed on 6 CpGs using Qiagen's Pyro Q-CpG software [22 ].