Quantifying GPX7 Expression in Gastric Carcinoma

Total RNA was isolated using the RNeasy Mini-kit (Qiagen, Valencia, CA USA). Single-stranded cDNA was subsequently synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA USA). Expression of GPX7 was evaluated for 45 gastric carcinomas and their matched normal gastric mucosae adjacent to cancers. The GPX7 primers (forward 5′-AACTGGTGTCGCTGGAGAAG-3′ and reverse 5′-AAACTGGTTGCAGGGGAAG-3′) were designed using the online software, Primer 3 (http://frodo.wi.mit.edu/). qRT-PCR was performed using an iCycler (Bio-Rad) with the threshold cycle number determined by use of iCycler software, version 3.0. Reactions were performed in triplicate and the threshold numbers were averaged. Results for the GPX7 gene were normalized to HPRT1 gene (forward 5′-TTGGAAAGGGTGTT TATTCCTCA-3′ and reverse 5′-TCCAGCAGGTCAGC AAAGAA-3′), which had minimal variation in all normal and tumor samples tested, and is therefore considered to be a reliable and stable reference gene for RT-PCR. Expression was calculated by use of the formula 2^(Rt–Et)/2^(Rn–En), as previously described [32 (link), 33 (link)]. For all of the primary gastric carcinoma samples, the gene was considered downregulated if the relative mRNA expression was ≤ 0.6, as compared to their matched normal samples.