Ma et al. Journal of Experimental & Clinical Cancer Research 2010, 29:85 http://www.jeccr.com/content/29/1/85 relation between BMP-2 and ovarian cancer remains unclear. This study was designed to determine the expression of BMP-2 and its receptors in epithelial ovar- ian cancer, benign ovarian tumors, and normal ovarian tissue and to analyze their influence on the five-year sur- vival rate and average survival time of ovarian cancer patients. Methods Samples RT-PCR samples: A total of 29 EOC patients, 32 benign ovarian tumor patients, and 10 patients with normal ovarian tissue were recruited from Shengjing Hospital, which is affiliated with China Medical University, between August 2005 and August 2007. Western blot samples: A total of 15 EOC patients, 15 benign ovarian tumor patients, and 10 patients with nor- mal ovarian tissue were recruited from Shengjing Hospi- tal, which is affiliated with China Medical University, between August 2005 and August 2007. Immunohistochemistry samples: One hundred paraf- fin-embedded specimens of EOC preserved at the Department of Pathology of Shengjing Hospital between January 1997 and August 2001 were included in this study. Specimens were examined for histological grade based on World Health Organization criteria. All EOC patients were grade II and grade III. The tumor stages were determined according to the International Federa- tion of Gynecology and Obstetrics (FIGO) with surgically and cytologically stage performed, all EOC patients had stage III and stage IV. All specimens were fixed with para- formaldehyde, embedded in paraffin, and prepared as serial slices of 4 μm in thickness. All experiment subjects had complete clinical patholog- ical data and were aged 20-72 years (mean: 50.36 ± 12.30), and there were no significant differences between age groups. No patients received radiotherapy, chemother- apy, biotherapy, or any other operation before surgery for the cancer. Maximal surgical cytoreduction is followed by the standard systemic chemotherapy for these patients. The pathological diagnosis was performed by experts at the Department of Pathology of Shengjing Hospital and the Fourth Hospital affiliated with China Medical Univer- sity. All samples and clinical data were obtained with the consent from all patients. Main reagents BMP-2 rabbit anti-human polyclonal antibody, SABC immunohistochemical kit, DAB color-developing reagent (Wuhan Boster, China); β-actin rat anti-human poly- clonal antibody, BMPRIA rabbit anti-human polyclonal antibody, BMPRIB rabbit anti-human polyclonal anti- body, BMPRII goat anti-human polyclonal antibody (Santa Cruz, USA); BMP-2, BMPRIA, BMPRIB, BMPRII Page 2 of 6 and internal reference β-actin primers (Shanghai Sangon, China); Trizol total RNA extraction reagent (Invitrogen, USA); RT kit and PCR amplification reagents (TaKaRa- Dalian, China); alkaline phosphatase-labeled goat anti- rabbit IgG, rabbit anti-goat IgG (Sigma, USA); Bradford protein kit (Nanjing Keygen, China); nitrocellulose filter (Shanghai Gene, China), β-naphthyl acid phosphate, O- dianisidine (tetrazotized; Sigma, USA). RT-PCR In accordance with the instructions for the Trizol total RNA extraction kit, total RNA was extracted from 100 mg specimens, and the ratio of OD260 and OD280 was 1.8- 2.0. The harvested RNA was diluted to a concentration of 1 μg/ul, packaged, and preserved at -70°C. The conditions for the first round of RT synthesis of cDNA were as fol- lows: 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min. PCR reaction conditions were as follows: for BMP-2, BMPRIA, BMPRII, and β-actin: 94°C for 2 min, 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s for a total of 30 cycles, then 72°C for 7 min; for BMPRIB: 94°C for 2 min, 94°C for 30 s, 53°C for 30 s, and 72°C for 45 s, for a total of 30 cycles, then for 72°C for 7 min. Primer sequences were as follows: BMP-2: 5'-CCAACCATGGATTCGTGGTG-3', 5'- GGTACAGCATCGAGATAGCA-3' BMPRIA: 5'-AATGGAGTAACCTTAGCACCAGAG-3', 5'-AGCTGAGTCCAGGAACCTGTAC-3' BMPRIB: 5'- GCAGCACAGACGGATATTGT-3', 5'- TTTCATGCCTCATCAACACT-3' BMPRII: 5'-ACGGGAGAGAAGACGAGCCT-3', 5'-CTAGATCAAGAGAGGGTTCG-3'; β-actin: 5'-GTGGGGCGCCCCAGGCACCA-3', 5'-CTCCTTAATGTCACGCACGATTTC-3' After 1.5% agarose gel electrophoresis with 1 μg/μl ethidium bromide dye, RT-PCR products were observed with a GIS-2020 gel scanning image analytical system. By using DNA Marker DL2000 as the standard molecular weight and β-actin as an internal reference, the ratio of BMP-2, BMPRIA, BMPRIB, BMPRII, and β-actin was calculated. RT-PCR products were semiquantitatively analyzed. Western blot In accordance with the instructions for the total protein extraction kit, total protein was extracted from 100 mg specimens. Protein concentrations were assayed by the Bradford method, and specimens were adjusted to the same protein concentration, packaged, and preserved at -