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.   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.