PCR was conducted with TaKaRa Ex Taq HS DNA polymerase in 50 μl reaction volumes. Primers (synthesized by Sangon Technology, Shanghai, China) used were including GAPDH (sense, 5′-ACGGATTTGGTCGTATTGGGCG-3′; antisense, 5′-CTCCTGGAAGATGGTGATGG-3′) with a product length of 197 bp and CD133 (sense, 5′-TTACGGCACTCTTCACCT-3′; antisense, 5′-TATTCCACAAGCAGCAAA-3′) with a product length of 172 bp. The reactions were conducted for GAPDH as the internal control under the following conditions: initial denaturing

step at 95°C for 1 min, 28 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by 72°C for 10 min; For CD133: initial denaturing step at 94°C for 2 min, 28 cycles at 94°C for 30 seconds, 51°C for 30 seconds, 72°C for 30 seconds, followed by 72°C for 10 min. according to the manufacturer’s instruction Five μl CD133 PCR and 2 μl of the products amplified by MyCycler™ Thermal Cycler (Bio-Red Laboratories, CA, USA) FK228 were separated on a 1.5% agarose gel (Gene Tech, Shanghai, China) by electrophoresis apparatus (Tunon, EpS 100, Shanghai Tian-neng Tech Co. Shanghai, China). Digital images to exposure the occurrence of CD133 mRNA as a white target strip were captured on a gel documentation system (UNIVERSAL HOOD II, Bio-Red Laboratories, Segrate, Milan, Italy). Imaging assessments

to measure the brightness scale value (BSV) of CD133 automatically from the write strip and to compared the relative ratio between CD133 strip and control strip were carried out by Quantity One 1-D analysis software (The Discoveries™ Quantity One SN-38 chemical structure 1-D Analysis Software Version 4.5, Bio-Red Laboratories, CA, USA.). Clinicopathological

assessments Clinicopathological parameters included gender, age, tumor size histological grade, invasion depth, lymph node metastasis, TNM stage, lymphatic vessel infiltration, vascular infiltration and metastatic lymph node ratio for CD133 protein and CD133 mRNA assessments respectively [13, 15], mainly according to UICC Avelestat (AZD9668) classification [15]. And Ki-67 LI was also used in the evaluation of CD133 mRNA expression. Prognostic analysis The deadline of follow-up for 99 patients was until November 2009, and the average survival time was 26.76 ± 17.02 months. A total of 9 cases (9.1% patients) lost in follow-up period. In this registered group, 39 cases died of the recurrence of gastric cancer, vascular diseases of brain or heart, or complications after surgery respectively. All patients in this group for survival assessment were divided as positive or negative subgroup of CD133 immunostaining. Statistics All statistical analyses were performed with the SPSS software version 13.0 (SPSS, Chicago, IL, USA). The correlations between expression of CD133 protein and clinicopathological parameters were assessed with the chi-squared test as a univariate analysis.