title: Transcriptome Profiling Reveals the Anticancer Mechanism of Polysaccharide from Gracilariopsis lemaneiformis
design: Cancer Cells were cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum(FBS) (Gibco, NY, U.S.A.), 100 units/ ml penicillin and 100 ug/ml streptomycin at 37℃ in a humidified incubator containing 5% CO2.  For PGL treatment and anticancer analysis, cells were seeded into a 6 well culture plate at a density of 5×105 cells/ well and treated with serial concentrations of PGL in a humidified atmosphere with 5% CO2 for 24, 48, and 72 h, respectively.  Total RNA was extracted by Trizol (Life Technologies, Inc. USA) and subjected to reverse transcription with random primer using the SuperScript III (Life Technologies, Inc. USA) to obtain cDNAs. Real-time quality PCR (RT-qPCR) was performed using a SYBR Green PCR Master Mix (Bestar, Germany) according to the manufacturer’s protocol. The primer sequences were used for Real-time PCR. Threshold cycles (Ct) were automatically calculated by the ABI step one plus (Applied Biosystems, USA). The PCR condition was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min. The intensity of each gene was normalized by GAPDH expression. According to the ΔΔCt method, differential expression was calculated as a ratio of the expression levels of target genes in PGL treated and untreated cells.