Davis et al. BMC Cancer 2011, 11:173 http://www.biomedcentral.com/1471-2407/11/173 Cells were cultured in 96-well plates at an initial den- sity of 3.0 x 103 per well. Each cell line was reverse trans- fected [25]with 25 nmol/l of siRNA and the appropriate transfection reagent diluted in Optimem® serum free medium (Invitrogen, Carlsbad, CA). HOSE 6.3 was trea- ted with 0.05 μl DharmaFECT 1 and JAM was treated with 0.1 μl DharmaFECT 3. All transfections were per- formed in triplicate and in three separate experiments. Transfection efficiency was determined by quantitative PCR (QPCR) whereby RNA was extracted from cells at 48 h post-transfection using the RNeasy Mini kit (Qia- gen, Valencia, CA, USA), in accordance with the manu- facturer’s instructions. Thereafter, cDNA was generated from 100 ng of RNA using Superscript III VILO (Invi- trogen, Carlsbad, CA), in accordance with the manufac- turer’s instructions. Expression of MAP2K4 was assessed using primers listed in Additional file 2, Table S2 on the LightCycler® 480 (Roche Diagnostics, Mannheim, Ger- many). PCR was performed using the SYBRgreen QPCR mix (Thermo Fisher Scientific, Lafayette, CO) and rela- tive MAP2K4 gene expression was determined according to the comparative CT (ΔΔCT) method: ΔΔCT = ΔCT MAP2K4 siRNA DNA - ΔCT Non-targeting siRNA DNA. ΔCT (threshold cycles) is the CT of the reference gene minus the CT of the target gene. Fold difference of MAP2K4 expression was calculated by 2-ΔΔCT. Hypox- anthine phosphoribosyltransferase 1 (HPRT1) was used as an endogenously expressed reference gene for the purpose of quantifying relative gene expression. Each QPCR was performed in triplicate using cDNA derived from three independent experiments. Cellular prolifera- tion was measured across a 7-day period according to the reduction of alamarBlue reagent (Invitrogen, Cals- bad, CA) following a 4 h incubation as described pre- viously [26]. Statistical Analysis Expression array files were imported into Partek Geno- mics Suite v 6.4 (Partek, St. Louis, MO) using default parameters including RMA normalization. These values were then used for statistical tests. Cox regression analy- sis was carried out in Partek Genomics Suite v 6.4. The results published here are in part based upon data gener- ated by The Cancer Genome Atlas pilot project estab- lished by the NCI and NHGRI. Information about TCGA and the investigators and institutions who constitute the TCGA research network can be found at http://cancer- genome.nih.gov. Other statistical analyses were carried out using GraphPad Prism software v5 (GraphPad Soft- ware, La Jolla, CA) and were considered significant at a P-value of less than 0.05. In assessing differences in pro- liferation, 2-way ANOVA was performed to interrogate time points as categorical variables. Page 3 of 7 Results and Discussion We previously reported MAP2K4 as a candidate tumor suppressor gene [18] after SNP array copy number and LOH data from 125 primary ovarian tumors identified four homozygous deletions with a minimal point of overlap of just 50 kb, which targeted two genes, MAP2K4 and hsa-mir-744. In the current study we decided to undertake further analysis of these genes to assess the frequency of gene inactivation by means other than deletion (the analysis of hsa-mir-744 will be described elsewhere). We analyzed exons 2-11 of MAP2K4 in 149 primary ovarian cancers using HRM and DNA sequencing. We identified two somatic altera- tions, a homozygous 16 bp deletion in exon 7 in a serous ovarian tumor (Figure 1), and a heterozygous 4 bp dele- tion in exon 3 in an endometrioid tumor. The 16 bp dele- tion in IC489 causes a frameshift alteration that would lead to protein termination 7 amino acids downstream (p.Asp263fs). The mutation represented more than 70% of the bases in the sequence trace, thus we consider it to be homozygous, correlating with the SNP array data for this tumor showing chromosome 17 LOH across the MAP2K4 locus. The 4 bp deletion at the start of exon 3 would cause protein termination two amino acids down- stream (p.Glu74fs) and is heterozygous in accordance with the lack of LOH at the locus in IC128 by SNP array. No known or novel polymorphisms were detected in the coding region of MAP2K4, which is consistent with the low number (n = 5) of reported coding variants (dbSNP build 130, http://www.ncbi.nlm.nih.gov/projects/ SNP/). Consequently, the failure to detect variants should not be attributed to a deficiency in the accuracy of the method, which has been successfully utilised to detect both novel and previously described polymorph- isms in a similar context [20,27]. Combining the detection of mutation and deletion data, the overall frequency of homozygous inactivation of MAP2K4 was 5.6% (5/89 samples analysed by both techniques). However, it was notable that cases with genetic changes were either of the high-grade serous (4/5) or grade 3 endometrioid (1/5) subtypes. As a pro- portion of these subtypes, inactivation of MAP2K4 was 9.8% (4/41) and 4.8% (1/21), respectively. We also ana- lyzed copy number data from The Cancer Genome Atlas (TCGA), a public data set of mostly high-grade serous ovarian tumors (http://cancergenome.nih.gov). In 157 SNP6.0 arrays, we identified a further 3 homozy- gous deletions, two of which specifically targeted only MAP2K4. TCGA has undertaken mutation screening by sequencing on 24 samples to date with no somatic alterations identified. We had previously observed a decrease in MAP2K4 RNA expression in samples with LOH at the locus [18].