Epitect methyl 2 dna restriction kit

Although all iPSC colonies were expanded to 6-well plates, 10 to 11 iPSC lines from each somatic source/reprogramming strategy combination (61 cell lines total) were selected based on round colony morphology and expanded for pluripotency characterization between passages 1–3. Each cell line was stained for SSEA1 (MC-480, Millipore, Temecula, CA) and alkaline phosphatase (Stemgent, Cambridge, MA) according to manufacturer’s instructions. In vivo teratoma formation assays were completed via subcutaneous injections of 500,000 L-MEF-iPSCs into the flank skin of immunocompromised nude mice [32 (link)]. Tissue sections of teratomas were subjected to hematoxylin and eosin staining (Biocare Medical). Immunohistochemistry was also performed on tissue sections of each teratoma using the ARK peroxidase kit (Dako) according to manufacturer’s instructions. mRNA was extracted from triplicate samples of selected iPSCs and mESC controls (R1, G4, CGR8) and cDNA was synthesized according to previously described protocols [27 (link)]. Quantitative real-time PCR (RT-PCR) on 10 ng cDNA was performed using primers listed in Supporting Information Table 1 (Integrated DNA Technologies) [27 (link)]. Methylation of promoter regions was quantified using the EpiTect Methyl II DNA Restriction Kit and EpiTect Methyl II PCR Primer Assays (Qiagen) according to manufacturer’s instructions.

Analysis of DNA methylation was performed using an EpiTect Methyl II PCR assay specific for the FLI1 promoter CpG island (Reference EPHS102855-1A; Qiagen). This assay is based on the differential cleavage of methylated versus unmethylated genomic DNA when using methylation sensitive or dependent restriction enzymes. Briefly, prior to performing the PCR reactions the DNAs were digested using the EpiTect Methyl II DNA Restriction Kit (Qiagen) according to the manufacturer’s instructions. The percentage of DNA that is fully methylated or fully unmethylated is determined by quantitative PCR (q-PCR) of digestion products. The q-PCR was set up by preparing individual reactions for each of the digestions following the protocol provided with the assay. Cycling was performed by the 7500 Real Time PCR System programmed according to the manufacturer’s instructions. The data were analyzed using the EpiTect Methyl II PCR Array Microsoft Excel template, available at www.sabiosciences.com/dna_methylation_data_analysis.php. This template automatically performs all ΔCt based calculations from the raw threshold cycle (Ct) values to determine gene specific DNA methylation status. The Excel template normalizes the Ct values of both digestions against mock digestion values to calculate and report the percentage of the DNA that is methylated and unmethylated.