Steponeplus amplification and detection system

Total RNA was isolated with Trizol from tumor as well as normal tissue samples collected from each patient and then purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Synthesis of cDNA was performed with 1 μg of total RNA from each sample using Quantiscript Reverse Transcriptase, Quantiscript RT-buffer and RT Primenr-mix of QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using in 96-well optical reaction plates (Applied Biosystems, Darmstadt, Germany) using a StepOnePlus amplification and detection system (Applied Biosystems). The real-time RT-qPCR reactions were prepared using SYBR® Select Master Mix (Life Technologies), and the following conditions were used: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The primers of target genes used for this analysis were Bim and PUMA, and GAPDH was used as the reference gene. The primer sequences are listed in Table S3. The gene copy numbers of Bim and PUMA were calculated by using a standard curve that was constructed using the OE33 cell line. The 2^−ΔΔCT method was used as a relative quantification strategy for qPCR data analysis. In total, 20 samples from Ile/Ile, 12 from Ile/Val and 6 from Val/Val genotype were used in this study.

Cells were collected from the inner layer of stomach from untreated mice (n = 10), RAN + lime and PRE + RAN + lime treated (n = 6 per point) for 180 and 260 days. Total RNA was extracted using an RNeasy Mini Kit (QIAGEN Co., Limburg, Netherlands). From 1 μg of total RNA, cDNA synthesis was performed using QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. For qPCR, cDNA was amplified using SYBER Green PCR mastermix according to the manufacture’s cycling condition for 40 cycle on a StepOnePlus amplification and detection system (Applied Biosystems). The primers of target genes used for this analysis were Mitotic arrest deficient 2 (Mad2) and Aurora A Kinase (AukA), and the primer sequences are listed in Table S1 (Supplementary section). Data were analysed using the delta-delta Ct method and plotted as fold change versus control (pl see Supplementary section).