Noerholm et al. BMC Cancer 2012, 12:22 http://www.biomedcentral.com/1471-2407/12/22 Page 7 of 11 Figure 4 Validation by qRT-PCR of down-regulated genes. Gene expression levels were normalized to the combined expression of GAPDH and 18S. Lines represent median values for the GBM and control samples, respectively. analysis, the RNA was linearly amplified and the same amount of RNA was hybridized to all arrays. For the qRT-PCR, the data was normalized to the house-keeping genes GAPDH and 18S. Interestingly, when the qRT- PCR data was normalized to the amount of RNA in the sample all genes initially found to be up-regulated by array analysis appeared to be down-regulated in serum microvesicles from GBM patients, as compared to nor- mal controls (Figure 5A). Inspection of the bioanalyzer profiles of the RNA samples used in this study sug- gested that the major contribution of the increased RNA amounts seemed to stem from a larger peak of small RNA migrating in the range from 25-300 nt on the bioanalyzer RNA chip (Figure 5B). Discussion Our findings demonstrate the feasibility of genome wide gene expression analysis of RNA extracted from the cell-free microvesicle fraction (exoRNA) of frozen bio- banked serum samples, and that clear differences can be observed between the exoRNA expression profiles from GBM patients and normal controls. The two major dif- ferences between exoRNA from GBM patients vs. nor- mal controls were found to be: 1) a significantly reduced level of mRNAs encoding ribosomal protein genes; and 2) an overall significant up-regulation of RNA amounts in the serum of GBM patients, which seems to be pri- marily due to a larger fraction < 300 nt and could not be attributed to any of the mRNAs investigated by qRT- PCR. ExoRNA from tumour microvesicles in serum will always be diluted into the background of exoRNA com- ing from normal non-malignant cells. Depending on the purpose of the study, normal exoRNA may also generate useful information. Previous studies have indicated that RNA expression patterns in blood cells can change as a response to tumours [7]. To look specifically at the genes dysregulated in the tumour, one would need to enrich the tumour specific microvesicle fraction before extracting the RNA. Microvesicles are shed by many different cell types into the blood stream, including circulating WBCs, pla- telets, endothelial cells, and dendritic cells [17]. Shed- ding has been shown to be higher from tumour cells in culture compared to normal fibroblasts [28] and tumour-derived microvesicle concentrations in serum increase as a function of increased malignancy [29,30]. However, microvesicles of tumour origin circulating in the blood are mixed with microvesicles from other cell Figure 5 Serum microvesicle RNA from patients with glioblastoma multiforme has an overabundance of RNA with size <500 nt. (A) Gene expression relative to the amount of microvesicle RNA present in serum of GBM patients and normal healthy controls. Bars represent the average expression of 16 genes as measured by qRT-PCR, normalized to the amount of exoRNA isolated from 1 mL of serum. The yield from GBM samples is higher than from controls, but this does not result in higher gene expression. (B) From the Bioanalyzer profiles it appears that the extra RNA in GBM serum is predominantly <300 nt. The plot is showing the mean ± SEM (N = 10 GBM and 10 controls). The non-visible parts of the profiles (>500 nt) were very similar for GBM and controls