Amplitag gold

In order to analyze CpG island hypermethylation of hBD-2, the methylation profiles were assessed using a methylation-specific PCR (MSP) method, similar to that reported by Herman et al (13 (link)). Briefly, DNA was extracted from the cultured cells with DNeasy Blood & Tissue kit (Qiagen, Stanford, CA, USA) according to the manufacturer’s protocol. The DNA was purified using a phenol/ethanol method. MSP distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated (but not methylated) cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA. The primer sequences are listed in Table I. PCR was performed using an amplification kit (AmpliTag Gold; Applied Biosystems) and a thermal cycler (Takara PCR Thermal Cycler MP; Takara, Osaka, Japan). Each PCR product was loaded directly onto non-denaturing 2% agar gels. As a positive control for methylation, CpGenome™ Universal Methylation DNA (Millipore, Billerica, MA, USA) was used.

DNA was isolated from 3- to 5-μm sections of FFPE tissues. After deparaffinization, the tumor tissue was processed with the QIAamp DNA Investigator Kit (Qiagen, Valencia, CA) according to the manufacturer´s instructions. Intronic primers were used to amplify exons 9, 11, 13 [51 (link)] and 17 [52 (link)] of KIT, exons 12 and 18 of PDGFRA [5 (link)] and exon 3 of CTNNB1 [53 (link)] by PCR in a reaction volume of 25 µL containing 5 µL of DNA, with 2,5 µL of buffer and MgCl₂ 25 mM and 0.5 µL of dNTP and 1.5 Units of ampliTag gold (Applied Biosystems, Foster City, Ca). PCR products were sequenced in F and R, after preheating the samples at 94° C for 6 minutes. The DNA was amplified over 40 cycles of 45 seconds of denaturation at 94° C; 1 minutes of annealing at 56° C and 1 minute of extension at 72° C, with an additional final extension step of 10 minutes. Negative controls were included in every set of amplifications. Bidirectional sequencing with specific primers was performed in an ABI 3130 xl genetic analyzer using the BigDye Terminator v3.1 kit (Applied Biosystems).