217004) according to the manufacturer’s instructions. Adult lung RNA was purchased from Ambion (Cat: AM6000). Immunostaining Antibodies against synthetic peptides from UPK3B, NKAIN4 and LRRN4 were ordered from IBM as a custom service. Immunostaining was carried out as described previously [29,30]. Results Identification of mesothelial markers in mouse Two sources of mesothelial tissue were dissected from 7 week old C57/BL6 mice. Total RNA was extracted from peritoneal mesothelium and a pool of pleural and pericardial mesothelium. Triplicate gene expression analysis was carried out using illumina microarrays on the mesothelium samples and unrelated tissue RNAs (adipose, bladder, heart, liver, lung, skeletal muscle). Data was normalized using the Lumi [19] package of bioconductor and differential gene expression between the mesothelium and other tissues was determined using B-statistics in the Limma [20] package. Peritoneal mesothelium or the pleural/pericardial pool was compared against each of the tissues separately. Only probes that showed significance (B-statistic .0 and at least 2 fold higher expression) in all comparisons (ie mesothelium vs heart, mesothe- lium vs liver etc.) were considered putative mesothelium specific markers. 460 probes (corresponding to 311 loci) passed this test as being over-expressed in at least one of the mesothelium sources compared to all the other tissues (Table 1, Table S3). Surprisingly only 48 of these were significant for both mesothelium sources. 223 were only over-expressed in the peritoneal mesothe- lium samples and 45 were only over-expressed in the pleural/ pericardial mesothelium samples. Of the 48 genes whose expression was enriched in both mesothelial sources seven were previously associated with the mesothelial lineage or mesothelioma (Aldh1a2 [31], Crip1 [32], Has1 [33], Prrx2 [34], Upk3b [35], Wnt2b [36], and Wt1 [37]). Interestingly Msln a previously reported marker of the mesothelial lineage was only found to be significantly more highly Table 1. Mesothelium enriched genes identified by microarray analysis. LRRN4 and UPK3B as Mesothelial Markers expressed in the peritoneal mesothelium and not in the pleural and pericardial sample. For the genes enriched in only one type of mesothelium we found separate classes of genes involved in immune response. In the peritoneal only list, CD34 was 4 fold higher expressed than in other tissues, and eight of the known defensins were also enriched (11–200 times that of any other tissue). However in the pleural + pericardial mesothelium there was enrichment for immunoglobulin heavy and light chains (2.3– 76 times that of any other tissue) suggesting possible B or plasma cell contamination. This suggests different sets of hematopoietic cells are associating with the mesothelium from different locations. To further investigate the gene sets we carried out gene ontology analysis using the GOstat tool [38]. There was no gene ontology enrichment for the genes highly expressed in the pleural/ pericardial mesothelium, but the list of genes expressed in both mesothelial tissues and the peritoneal only samples were enriched for genes involved in cell adhesion and extracellular space. The peritoneal set was also enriched for genes involved in develop- mental processes and multicellularity (Table S4). qRTPCR testing of candidate markers in selected mouse tissues To further focus on biomarkers of mesothelium we chose a set of 36 candidates for further confirmation by qRTPCR which were likely to be secreted, transmembrane proteins or novel. This included 11 of the genes enriched in both mesothelium sources, 20 genes only enriched in peritoneal mesothelium and 5 genes only enriched in the pleural and pericardial mesothelium (Table S1). The mesothelium specific expression of the 36 candidate biomarkers was then tested by qRTPCR across a broader selection of tissue RNAs (pleural + pericardial mesothelium, peritoneal mesothelium, lung, adipose, heart, liver, brain, kidney, thymus, spleen, intestine, testis) and 14 candidates that were still shown to be meosthelial specific were further tested against an additional 5 other tissues (bladder, skeletal muscle, colon, uterus, ovary) and also against mesothelium from female mice to confirm there were no sex specific differences. This secondary screening Both sources Peritoneal only Mesothelium enriched genes 2010001M06Rik , 2010107G12Rik , 4930415N12Rik , 4930455G09Rik , A730017C20Rik , A730046J19Rik , Acot11 , Aldh1a2 , Anxa9 , Aplp1 , Ccdc109b , Cdon , Celf4 , Chst4 , Cldn15 , Crip1 , Cybrd1 , D930030F02Rik , Dcaf12l2 , Fam70a , Flrt2 , Gpm6a , Has1 , Ildr2 , Isyna1 , Lgals7 , Lrrn4 , Nkain4 , Nxnl2 , Pkhd1l1 , Prrx2 , Rpp25 , Rspo1 , Saa3 , Serpinb2 , Sez6l , Slitrk4 , Smpd3 , Spns3 , Sulf1 , Tmem114 , Tnfrsf11b , Trem2 , Tubb2b , Upk3b , Vit , Wnt2b , Wt1 1110012D08Rik , 1110064A23Rik , 1500015O10Rik , 1700023F06Rik , 2410039M03Rik , 2600006K01Rik , 2900073C17Rik , 4930533K18Rik , 5430435G22Rik , 5730409K12Rik , 6330406I15Rik , 7330423F06Rik , 9530077C05Rik , Abcg4 , Accn1 , Adamts19 , Adamts2 , Aebp1 , Agap1 , AI661453 , AI747448 , Ak5 , Alox15 , Anks6 , Anln , Arhgap29 , AY761184 , AY761185 , B3gnt9-ps , Bicc1 , C2 , C330020E22Rik , Cacna1g , Capn6 , Casp12 , Ccdc80 , Ccin , Ccrl1 , Cd109 , Cd34 , Cdh11 , Cdh3 , Cercam , Cgnl1 , Chrd , Clca6 , Clec11a , Clec4d , Clps , Col6a1 , Col6a2 , Col6a5 , Colec12 , Cox6b2 , Cplx2 , Cpt1c , Cpz , Crb2 , Creb3l1 , Csrp2 , Dbn1 , Dcn , Ddr1 , Defa17 , Defa20 , Defa22 , Defa23 , Defa3 , Defa5 , Defa6 , Defa- rs1 , Dgkb , Dhrs9 , Disp1 , Dlc1 , Dlk1 , Dmrt3 , Doc2b , E330013P04Rik , Ecm1 , Efemp1 , Emilin2 , Eno2 , Enox1 , Enpp2 , Ephb6 , Espn , Etv1 , Fam114a1 , Fam180a , Fam184a , Fbln2 , Fbn1 , Fez1 , Fgf1 , Fgf18 , Fgf5 , Fgf9 , Fkbp10 , Fkbp14 , Fkbp9 , Flrt1 , Fosb , Gabrb1 , Gabrq , Galns , Galntl2 , Garnl3 , Gas6 , Ghrh , Gjb3 , Glb1l , Glt8d2 , Gm12680 , Gng4 , Gpr85 , Gria3 , Guca1a , Gxylt2 , Hif3a , Htra1 , Ifitm1 , Igfbp6 , Ikbip , Il31ra , Il7 , Ildr2 , Isl1 , Islr , Itln1 , Kcnab1 , Kcnip3 , Kirrel , Krt76 , Lamc3 , Leprel2 , Lgals1 , Lgals2 , Loxl1 , Loxl2 , Lrpap1 , Ltbp3 , Man2b2 , Mapk4 , Mdk , Mfap5 , Miat , Mmp16 , Mmp23 , Mmp24 , Msln , Mtmr11 , Muc13 , Muc16 , Muc2 , Nbl1 , Ndrg1 , Neo1 , Nfkbiz , Nid2 , Ninl , Nkx2-3 , Nlgn1 , Nrxn2 , Nxph1 , Oasl2 , Olfml2b , Olfr1036 , Pcolce , Penk , Piwil2 , Plat , Plekha4 , Prg4 , Ptgis , Rassf2 , Rcn3 , Reg3b , Rnf112 , Rtn1 , Scg5 , Scrn1 , Selp , Selv , Sema5a , Serpinh1 , Sertad4 , Sfxn3 , Sgsm2 , Slc1a7 , Slc23a3 , Smpd3 , Smpdl3b , Snord123 , Sorcs1 , Sox8 , Spink3 , Ssbp3 , Stag3 , Steap2 , Stra6 , Sulf1 , Sv2a , Svs5 , Tmem119 , Tmem151a , Tmem98 , Tnxb , Trim9 , Tro , Ttll12 , Ttyh1 , Tulp2 , Upk3b , Vasn , Vat1l , Vmn2r26 , Wasf1 , Wnt6 , Zbtb7c , Zbtb8a , Zfp185 , Zg16 Pleural + pericardial 1190002F15Rik , 2810417H13Rik , A530032D15Rik , A530050N04Rik , AB124611 , AI324046 , Aurkb , B230209E15Rik , Bfsp2 , Btg3 , Calcb , Csprs , only Cxcl13 , Cybrd1 , Dntt , Expi , Fcna , Gng13 , Gpr176 , Hist1h2an , Igfbp5 , Kif22 , Oosp1 , Plac1l , Prss16 , Rag1 , Rilpl2 , Sdc3 , Sema3d , Serpina3b , Slc2a6 , Spo11 , Stac2 , Tbata , Tm4sf5 , Tnfrsf17 , Uhrf1 , Xpnpep2 , Zbtb32 Note: Gene names in bold have been previously associated with mesothelioma or the mesothelial lineage. doi:10.1371/journal.pone.0025391.t001 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25391