Magna pure lc dna isolation system

HUMARA analysis was performed using peripheral blood mononuclear cells (MNC) obtained at various time points before and during TKI therapy, following published techniques [41 (link)]. In brief, DNA was isolated from MNC using the MagNA Pure LC DNA isolation system (Roche) according to the manufacturer's recommendations. The X-chromosome inactivation (XCI) pattern was determined by PCR analysis of the polymorphic CAG-repeat region in the HUMARA gene which is located next to an HpaII restriction site. Genomic DNA (~250 ng) was digested with HpaII for 19 hours at 37°C. Then, aliquots of undigested and HpaII-digested DNA were subjected to PCR using published primer sequences [41 (link)]. Amplicons were analyzed on an Applied Biosystems (ABI) 3130xl genetic analyser (Foster City, CA, USA) using the Gene Scan 3.7 (ABI) software. For quantification, the areas under the peaks of the two alleles from the undigested and digested DNA were determined and the ratio of the peak area of both alleles (X1, X2) was calculated (in summary 100%). The degree of X-chromosome inactivation is presented as percent of the smaller peak (X2) in relation to the larger peak (X1). Undigested female DNA usually shows 2 peaks of similar height (in 90% of females).

Genomic DNA was isolated from blood samples using the MagNA Pure LC DNA Isolation system (Roche Molecular Biochemicals, Penzberg, Germany). The rs7574865 polymorphism of STAT4 was genotyped using the TaqMan SNP genotyping assay (Applied Biosystems, Foster City, California, USA; Part number: C__29882391_10). The polymerase chain reaction (PCR) assay was carried out according to the manufacturer’s recommendations. After PCR, the genotype of each sample was attributed automatically by measuring allele-specific fluorescence on a StepOnePlus Real-Time PCR System (Applied Biosystems).