Quantification of mRNA expression was performed in triplicate using quantitative reverse transcription polymerase chain reaction (PCR) with the following primers: Forward, 5′-CTCCCATCTCAACTCCCTGA-3′ and reverse, 5′ACCAGCTTCCTGAACAGCTC-3′, for P2X7. Specific TaqMan® miRNA Assays (Applied Biosystems Life Technologies, Foster City, CA, USA) were used for Let-7 g, miR-21, miR-205 and RNU6B according to the manufacturer’s instructions. The threshold cycle (Ct) and baselines were determined using manual settings, where Ct was set in the linear phase of the amplification and baseline in the initial cycles of PCR. Expression was calculated by relative quantification and fold expression changes were determined by the 2−ΔΔCT method using the DataAssist software (Applied Biosystems Life Technologies).
Mutational analysis was also performed for all samples. The mutational status of the codons 12–13 of the K-Ras gene was analyzed by pyrosequencing using the anti-epidermal growth factor receptor (EGFR) MoAb response® kit (K-Ras status) (Diatech Pharmacogenetics, Jesi, Italy) according to the manufacturer’s instructions.
PCR-single stranded conformation polymorphism and sequencing analysis were used for EGFR genotyping in exons 18–21, as previously described (14 (link)).
Mutational analysis was also performed for all samples. The mutational status of the codons 12–13 of the K-Ras gene was analyzed by pyrosequencing using the anti-epidermal growth factor receptor (EGFR) MoAb response® kit (K-Ras status) (Diatech Pharmacogenetics, Jesi, Italy) according to the manufacturer’s instructions.
PCR-single stranded conformation polymorphism and sequencing analysis were used for EGFR genotyping in exons 18–21, as previously described (14 (link)).
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