Qian et al. Journal of Experimental & Clinical Cancer Research 2010, 29:111 http://www.jeccr.com/content/29/1/111 Page 2 of 5 promoter was methylated in AML and MDS. The sup- pressed GRAF expression could be restored in leukemic cell lines by treatment with a demethyating agent and an inhibitor of histone deacytylases. However, the expression level of GRAF gene has not yet been studied in leukemia. We established the real-time quantitative polymerase chain reaction (RQ-PCR) assay with Eva- Green dye and examined the expression level of GRAF mRNA in myeloid malignancies. Materials and methods Patients and samples The bone marrow mononuclear cells (BMNCs) from 94 patients with myeloid malignancies, including 72 AML, 7 MDS and 15 chronic myeloid leukemia (CML), were studied. The diagnosis and classification of AML and MDS patients were based on the French-American- British (FAB) and World Health Organization (WHO) criteria (blast ≥ 20%) combined to immunophenotyping and cytogenetic analysis [11-15]: among AML, 12 cases of M1, 23 cases of M2, 13 cases of M3, 18 cases of M4, 5 cases of M5, 1 case of M6; among MDS, 1 case of refractory anemia with ring sideroblasts (RARS), 2 cases of refractory cytopenia with multilineage dysplasia (RCMD), 3 cases of refractory anemia with excess blasts-1 (RAEB-1), 1 case of RAEB-2. The diagnosis of CML was established according to the conventional cri- teria [16]: 10 cases at chronic phase (CP), 5 cases at blast crisis (BC). The clinical characteristics of patients were listed in Table 1. Karyotypes were analyzed using conventional R-banding method. Karyotype risk in AML and MDS was classified according to the reported studies [15,17]. t(15;17) was also included in the group of low risk. BMNCs, collected from 3 donors of bone marrow trans- plantation, 5 patients with immune thrombocytopenia (ITP), and 13 with iron deficiency anemia (IDA), were used as controls. Immunophenotyping studies Erythrocyte-lysed whole BM samples from 61 AML patients were analyzed by flow cytometry using a panel of MoAbs in triple stainings [phycoerythrin (PE)/fluores- cein isothiocyanate (FITC)/Peridin chlorophyll (PerCP)]: IgG1-FITC; IgG2a-PE; CD2-PE; CD4-FITC; CD7-FITC; CD10-FITC; CD11b-PE; CD13-PE; CD19-PE; CD38-PE; CD45-PerCP; CD117-PE; HLA-DR-FITC (Becton Dickinson, USA); CD14-PE; CD22-FITC; CD33-FITC; CD34-FITC; CD36-FITC (Beckman Coulter, USA). Data acquisition and analysis were performed on a FACScali- bur flow cytometer (Becton Dickinson) using Cell-Quest software. Identification of leukemic cells was performed using CD45 intensity versus SSC dot plots. Antigen expression was considered to be positive when the per- centage of positive leukemic cells was equal or greater than 20%. Preparation of RNA and cDNA synthesis BMNCs were separated using Lymphoprep and lysed with Trizol (In Vitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two micrograms of total RNA was reverse transcribed to cDNA in a total reaction volume of 40 μl containing 5× buffer, dNTPs 10 mM each, random hexamers 10 μM, RNAsin 80 units and 200 units of MMLV reverse transcriptase (MBI Fermentas, USA). Samples were incubated for 10 min at 25°C, 60 min at 42°C, and then stored at -20°C. RQ-PCR RQ-PCR was performed using EvaGreen dye (BIO- TIUM, Hayward, CA, USA) on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA). Real-time fluorescent data were collected and analyzed with SDS 1.3 software (Applied Biosystems, Foster City, CA, USA). The baseline fluorescence intensities were fixed at cycles 6-15 by default and 0.01 was set as the threshold to determine the cycle threshold (CT) value. The Table 1 clinical and laboratory features of patients with myeloid malignancies 23.51(0.01-157.42)c 10.20(0.25-45.90)b WBC, white blood cells; aMedian (range); bP < 0.001, compared with control; cP = 0.030, compared with control; CML 52(11-75) 8/7 83.4(2.8-168.7) 91(50-134) 200(20-850) MDS 63(39-85) 5/2 3.6(1.6-12.2) 64(46-91) 50(10-926) 3 3 1 Parameter Age, median (range) (years)a Sex (male/female) WBC (×109/l)a Haemoglobin (g/dl)a Platelet count (×109/l)a Cytogenetics Good Intermediate Poor CD34(+/-) GRAF levela AML 54(2-86) 44/28 7.5(0.3-203.6) 71(24-123) 40(3-447) 22 35 8 35/26 3.88(0.01-169.75)b