Noerholm et al. BMC Cancer 2012, 12:22 http://www.biomedcentral.com/1471-2407/12/22 Page 6 of 11 Table 1 Down-and up-regulated genes passing the criteria described in ?“?Results?”? (Continued) 20 21 22 23 24 25 TNRC4 AK098372 TNRC6B HOXA4 IL26 THC2718728 1.72 1.94 1.79 1.58 1.66 1.73 Homo sapiens trinucleotide repeat containing 4 (TNRC4), mRNA [NM_007185] Homo sapiens cDNA FLJ25506 fis, clone CBR05185. [AK098372] Homo sapiens trinucleotide repeat containing 6B (TNRC6B), transcript variant 1, mRNA [NM_015088] Homo sapiens homeobox A4 (HOXA4), mRNA [NM_002141] Homo sapiens interleukin 26 (IL26), mRNA [NM_018402] Unknown “Fold” is calculated as the median value of the fold change in the three described normalization procedures those of platelets (GSE11524) and PBMCs (GSE22224) from normal controls to derive a list of “GBM-like” genes highly expressed in GBM, but low or absent in platelets and PBMCs and similarly a list of “blood cell- like” genes highly expressed in platelets and PBMCs. When we compared the “GBM-like” genes to the genes found to be up-regulated in GBM patient blood micro- vesicle RNA in our own dataset, we found no overlap to support GBM tumour origin of these RNAs. On the other hand, many of the “blood cell-like” genes were the same as those found in our study to be down-regulated in GBM patients relative to normal controls (data not shown). In selecting genes for further qPCR validation we also considered whether genes were represented in the GO clusters and whether they were genes previously deter- mined to have cancer association. The following genes were chosen for validation studies by qRT-PCR: RPL11, RPS12, TMSL3 and B2M as expected down-regulated genes and EGFR, ERBB2, SLITRK4, HOXA4, METT5D1, CV575560, TNXB, ALUY, 7SL and THC2718728 as pos- sible up-regulated genes. For normalization of the qRT- PCR data we chose GAPDH, which appeared to be stable in our exoRNA samples judging from the array data, as well as 18S rRNA. The qRT-PCR validation was performed on a different set of GBM patients (N = 10) and controls (N = 10) than the array data. We were able to confirm the down-regulation of genes associated with ribosome production RPL11, RPS12, TMSL3 and B2M observed in the arrays as illustrated in Figure 4, but the expected up-regulated genes did not show any signifi- cant increase relative to controls by qRT-PCR (data not shown). RNA was extracted from equal volumes of serum (1 mL) from GBM patients and normal controls. The exoRNA content of the GBM patient serum sam- ples used for qRT-PCR validation in this study, (30.0 +/- 8.3 ng/mL serum; N = 10) was significantly higher (p < 0.001) than in the normal control serum samples (8.8 +/- 2.2 ng/mL serum; N = 10). For the microarray Figure 2 Analysis of the 400 most dysregulated genes. The 200 most down- and up-regulated genes, respectively, with p < 0.05 in all three normalizations after correction for False Discovery Rate were used (see Additional file 2: Table S1 and Table S2). A) Cluster analysis, B) PCA plot. Figure 3 Volcano plot of the False Discovery Rate (FDR) corrected p-values from a t-test between the two sample groups after background subtraction and quartile normalization. Genes above the horizontal dashed line have p < 0.05 after FDR correction. It is evident that substantially more genes are significantly down-regulated (121 genes in upper left corner, > 2-fold) than up-regulated (24 genes in upper right corner, > 2-fold).