For definitive diagnosis, immunohistochemical staining of biopsy material for epithelial cell adhesion molecule (EPCAM) and Vimentin (VIM) have been used to discriminate mesothelioma from other lung cancers [9,10], however more recently a microRNA based test with a higher accuracy has been developed and made available to doctors [11]. These are important for determining treatment however none of these are used for early detection. Recently a kit (MESOMARK [12,13]) for detection of soluble mesothelin related peptides in patients’ blood has made it to market. This ELISA based kit is currently the most specific and sensitive to market [14]. Mesothelin was originally identified as megakaryocyte potentiating factor (MPF), and later termed mesothelin when it was shown that an antibody against it reacted with ovarian cancers and malignant melanomas [15,16] and as early as 1999 it was shown that mesothelin related peptides could be detected in the serum of ovarian cancer patients [17]. Mesothelin is produced as a transmembrane protein which is then cleaved to release (MPF). Considering the poor prognosis and the long latent period before diagnosis there is both a need for earlier diagnosis and a real opportunity to develop earlier tests (10–20 years window). In general earlier diagnosis means improved likelihood of survival [18]. With this in mind we set out to identify additional mesothelial lineage markers using a comparative functional genomics approach. Gene expression profiling of dissected mouse mesothe- lium and other tissues was used to identify candidate biomarkers enriched in mesothelium. We then went on to filter these lists by first focusing on candidate biomarkers that were likely to be secreted or transmembrane proteins and then validating the specificity of the markers by quantitative in-situ hybridization in mouse tissues and real time PCR (qRTPCR) across a panel of human cancer cell lines and normal lung cells and mesothelial cell RNAs. Materials and Methods Ethics Statement All animal work was carried out according to institutional and national ethical guidelines, and approved by the Yokohama Animal Experiment Review Committee, Plan19-002. Similarly all human work was carried out according to institutional and national ethical guidelines. The human mesothelioma sample used in the immunohistochemistry was made available anonymously from the autopsy material collection at the Department of Pathology and Oncology, Juntendo University School of Medi- cine. The human primary mesothelial cells were purchased from Zenbio. The human primary cells were purchased from Zenbio, Sciencell, Cell Applications and Lonza. Animal and Tissue sources The tissues of 7-week-old male mice on C57BL/6 backgrounds were used for RNA preparation and for ISH. For RNA preparation, mice were anesthetized, then sacrified, then their organs were removed or harvested and immediately washed with Phosphate-Buffered Saline (Invitrogen) and transferred into RNA later (Ambion). Two sets of mesothelial tissue were prepared from 200 mice. Peritoneal mesothelium was prepared as one source and pleural and pericardial mesothelium were pooled as the second source. Lung, liver, brain, heart, skeletal muscle, subcutaneous adipose, testis, bladder and kidney tissues were also dissected as required for RNA extraction. For ISH, Mice were anesthetized with diethyl ether and transcardially perfused with PFA solution (4% paraformaldehyde, 4% sucrose in sodium phosphate buffer pH 7.5) and then with LRRN4 and UPK3B as Mesothelial Markers Bouin’s solution (71% saturated picric acid [v/v], 24% formalin [v/v], and 5% acetic acid [v/v], adjusted to pH 4.0 with sodium hydroxide). The brains were then removed, fixed with original Bouin’s solution for 3 days, and embedded in paraffin. All procedures involving animals and their care were carried out in accordance with the directives of RIKEN’s Institutional Animal Care and Use Committee. RNA extraction Total RNA was purified using the RNeasy mini kit (Qiagen) and RNeasy Lipid Tissue Midi kit (Qiagen) according to manufactur- er’s instructions. The quality of the extracted RNA was confirmed with Agilent 2100 Bioanalyzer. Microarray Five hundred nanogram of total RNA was amplified using the Illumina TotalPrep RNA Amprification Kit (Ambion), according to manufacturer’s instructions. cRNA was hybridized to Illumina Mouse Sentrix-6 Expression BeadChip Ver.1, according to standard Illumina protocols. Chips scans were processed using Illumina BeadScan and BeadStudio software packages and summarized data was generated in BeadStudio (version 3.1). Data was quantile normalized using Lumi and B-statistics were calculated using Limma (both R packages in Bioconductor) [19,20]. Meosthelial tissue gene expression was compared pairwise against each other tissue, and only genes where the B-statistic was greater than 0 for all cases and the average expression in the mesothelial tissue was greater than 2 fold for all other tissues was the expression considered mesotheial specific. All microarray data is MIAME compliant and the raw data has been deposited in a MIAME compliant database (GEO (ID pending)). qRTPCR qRTPCR was carried out as described previously [21]. Primers used are listed in Table S1. Primers for human MSLN were from Jirsova et al [22], all other primers were designed in house. In situ hybridization (ISH) ISH was carried out using PCR generated probes with FANTOM1 and FANTOM2 cDNA clones or genomic DNA as templates (Table S2). ISH was carried out as described previously [23]. Human total RNA samples Primary cells or RNA were purchased: Mesothelial cells– (Zenbio, Cat: F-MES-F), small airway epithelial cells (Lonza, Cat: CC-2547), lung fibroblasts (Cell applications, Cat: CA506-R10f), alveolar epithelial cells (Sciencell, Cat: SC3205), bronchial epithelial cells (Sciencell, Cat: SC3215), bronchial smooth muscle cells (Sciencell, Cat: SC3405), tracheal smooth muscle cells (Sciencell, Cat: SC3415), retinal pigment epithelial cells (Sciencell, Cat: SC6545) and urothelial cells (Sciencell, Cat: SC4325). The cell lines (ACC-MESO-1, ACC-MESO-4 [24], LC-1F, EBC-1, LU65, Lu99, PC-14, A549, Lu-134-A, LK-2, IA-5, T3M-10, HeLa, OS-RC-2, JMSU1, TGBC2TKB, TGBC18TKB, A431, Kato III and WERI-Rb-1) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The additional mesothelioma cell lines were purchased from the American type culture collection (NCI-H28, NCI-H226, NCI- H2052, NCI-H2452 [25]) and the European Collection of Cell Cultures (Mero-14, Mero-25, Mero-41 [26], Mero-48a, Mero-82, Mero-83, Mero-84, Mero-95 [27], No36, ONE58 [28]). Total RNA was prepared using QIAGEN miRNeasy columns (Cat: PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25391