Abi prism 9700

Tissue samples submitted for mutational analysis were obtained through biopsy or surgical resection. Genomic DNA of the tumor specimen was extracted using a microdissection method based on the manufacturer's protocols. The nucleotide sequences encoding the kinase domain (exons 18–24) of EGFR were amplified via a quantitative real-time polymerase chain reaction (PCR)-based method (qPCR). The presence of an appropriate PCR product was confirmed by resolving the PCR products on a 2% agarose gel. After purification, corresponding fragments on the gel were sequenced in both sense and antisense directions using an ABI PRISM® 9700 and ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA). The sequenced data using SeqScape (Applied Biosystems) were analyzed and compared with the archived human sequence of EGFR (GenBank accession no. NG_007726.1), to identify the mutation. Of the 115 patients who were tested for somatic mutations, 64 patients were mutant-type of EGFR, whereas 51 patients tested negatively for the EGFR mutation (wild-type of EGFR).

Genomic DNA from cases and from controls was isolated from EDTA blood samples using standard salting out procedure [18] (link).
Genotyping of the FTO variant rs9939609 was performed by Real-time TaqMan Allelic Discrimination Assay using pre-designed primers obtained from Applied Biosystems (Pre-designed TaqMan SNP Genotyping Assays, 7500 Real time PCR System, Applied Biosystems, Assay ID C__30090620_10) on ABI PRISM 9700 platform (Applied Biosystems).
We genotyped 874 completed suicide subjects (+68 undetermined samples, 93% call rate). As the call rate was low, we purified 43 undetermined samples containing enough DNA using Agencourt AMPure XP (Beckman Coulter) which allowed to successfully genotype additional 38 samples bringing the total number of samples successfully genotyped to 912 (call rate 97%). Among controls 733 subjects were successfully typed (14 undetermined genotypes, 98% call rate).