To begin, transfect the stomach adenocarcinoma cells with TMEM200A siRNA and incubate for two days. Then, discard the cell culture medium from the wells and gently wash the cells two times with one milliliter of PBS. Using an RNA isolation kit, isolate the total RNA of the cells.
Estimate the RNA concentration and synthesize cDNA with one microgram of RNA using a cDNA synthesis kit, following the manufacturer's instructions. Now set up quantitative RTPCR on tenfold dilutions of cDNA in a 10 microliter reaction mix. Add primers and quantitative PCR master mix using the realtime PCR assay system.
Use the quantitative PCR reaction conditions shown here and determine the relative expression of each gene using the double delta CT technique. The HGC-27 cell line dramatically over expressed TMEM200A protein and mRNA transcript compared to the GES-1 cell line. The differential mRNA expression of TMEM200A in human gastric cancer cells, SGC-7901, is higher than in GES-1 cells.
