Qian et al. Journal of Experimental & Clinical Cancer Research 2010, 29:111 http://www.jeccr.com/content/29/1/111 Page 3 of 5 primers of GRAF and housekeeping gene ABL were designed against GenBank-published sequences (NM_015071 and NM_14752) with the software Primer Express 2.0 (Applied Biosystems, Foster City, CA, USA). The primer sequences are as follows: GRAF forward 5′- ATTCCAGCAGCAGCTTACA-3′, reverse 5′-GATGAG GTGGGCA TAGGG-3′, ABL forward 5′-TCCTCCA GCTGTTATCTGGAAGA-3′, reverse 5′-TCCAACGA GCGGCTTCAC-3′, with expected PCR products of 166 bp and 118 bp, respectively. PCR was performed in a final volume of 25 μl, containing 100 ng of cDNA, 0.2 mM of dNTP, 4 mM of MgCl2, 0.4 μM of primers, 1.2 μl of EvaGreen, 1.0 U of Taq DNA Polymerase (MBI Fermentas, USA). Amplification consisted of an initial denaturation step of 94°C for 4 min followed by 40 cycles of a denaturation step at 94°C for 30 s, an anneal- ing step at 62°C for 30 s, an extension step of 72°C for 30 s, and an fluorescence collection step at 82°C for 30 s, followed by a final extension of 72°C for 10 min. Ster- ile H2O without cDNA used as no-template control (NTC) in each assay. The copies of GRAF and ABL mRNA were calculated automatically by the software. The relative amount of GRAF was normalized using the following formula: NGRAF = (copies of GRAF/copies of ABL) × 100. Amplified RQ-PCR products from three samples were sequenced (Shanghai GeneCore Bio- Technologies Co., Ltd., China). Statistical analyses Statistics was performed using the SPSS 13.0 software package (SPSS, Chicago, IL). The Kruskal-Wallis test (multiple groups) and Mann-Whitney U-test (two groups) were employed to compare the difference between patient groups and controls. The correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups was calculated by Spearman’s rho correlation analyses. A P-value < 0.05 was considered significant. Results GRAF expression in controls and AML patients The level of GRAF transcript in controls was 14.49- 126.85 (median 56.04). The significantly decreased level of GRAF transcript was observed in different myeloid malignancies (Table 1, Figure 1). There was no correlation between GRAF mRNA amount and the sex, age, peripheral white blood cell count, hemoglobin level, and platelet count (P > 0.05). The association of GRAF levels with cytogenetic abnormalities or CD34 antigen expression was also not observed in AML patients (P > 0.05). Within AML, there was no differ- ence in the level of GRAF transcript among different FAB subtypes (P > 0.05). Figure 1 Scatterplot showing varying levels of GRAF transcript in patients with different myeloid malignancies and controls. GRAF expression in CML patients The median levels of GRAF transcript in CML patients at CP and BC were 46.82 (1.08-157.42) and 10.69 (0.01- 23.51), respectively (Figure 2). There was no difference in GRAF transcript amount between CML patients at CP and controls (P > 0.05). However, the amount of GRAF mRNA in CML at BC was significantly lower than that in cases at CP and that in controls (P = 0.028 and <0.001, respectively). GRAF expression in MDS patients Among MDS patients, three cases were identified with deletions of 5q (5q-) (Table 2). The level of GRAF Figure 2 Expression level of GRAF transcript in CML.