Noerholm et al. BMC Cancer 2012, 12:22 http://www.biomedcentral.com/1471-2407/12/22 on RNA derived from blood samples to differentiate between healthy controls and patients with certain types of cancer [5-8], to classify separate patient populations [9] or to predict clinical outcome [10]. The ability to conduct nucleic acid expression profiling assays on a blood sample rather than on tumours has a wide range of implications for patient welfare, including the ability to conduct longitudinal disease monitoring in situations where tumour tissue is not easily accessible or one is trying to sample metastatic cancer. Because the blood harbors nucleic acid of both tumour and non-tumour origin, it is possible that this approach may capture not only direct nucleic acid changes seen in the tumour cells, but also a component of the host response to the presence of tumour. For example, studies so far have reported on RNA extracted from Peripheral Blood Mononuclear Cells (PBMC) or other fractions of circu- lating blood cells where changes in the cell RNA profile appears to represent the host’s response to the malig- nancy [5] rather than the tumour itself. Different groups have isolated RNA from circulating tumour cells [11] and from cell-free body fluids [12]. Given the short half- life of unprotected RNA in serum [13], it is likely that most of the cell-free RNA is protected in the exosomes/ microvesicle fraction or in the case of microRNAs (miR- NAs) by protein complexes in the blood [14,15]. Microvesicles are very stable and can protect cell-free RNA stored in the freezer for many years. This is a great advantage compared to analyzing circulating tumour cells where the blood needs to be processed within hours of collection. In addition, circulating tumour cells have not yet been described in glioma patients [16]. In this study, full microarray analysis was carried out on mRNA isolated from serum microvesicles (including exosomes and other shedding microvesicles [17]) from GBM patients and controls to test the hypothesis that this mRNA could be used to reflect tumour-associated changes in the exosomal/microvesicle fraction of serum RNA. RNA species showing differential expression were chosen for quantitative reverse transcriptase (qRT-PCR) validation. This study is the first to report the ability to differentiate GBM patients from normal controls based on a gene expression blood test and the first to report differential expression analysis using RNA extracted from exosomes/microvesicles isolated from clinical patient serum samples, as compared to controls. Methods Clinical samples Blood samples from patients diagnosed with de-novo pri- mary GBM were collected immediately prior to surgery (before opening of the dura mater) into a BD Vacutainer SST (#367985) at Massachusetts General Hospital (MGH). Patients were following standard of care at Page 2 of 11 MGH, including fasting prior to surgery and most of the patients were treated with steroids to alleviate vasogenic edema and pain. Blood from normal healthy controls was collected from de-identified volunteers recruited at the MGH blood bank. All samples were collected with informed consent according to the appropriate protocols approved by the Institutional Review Board at MGH. The blood was left to clot for 30 min at room temperature (r. t.) and serum was isolated, according to manufacturer’s recommendations within 2 h of collection. Serum was fil- tered by slowly passing it through a 0.8 μm syringe filter (Millopore, Billerica, MA, USA) and aliquoted into 1.8 mL cryotubes (Fisher Scientific, Waltham, MA, USA) and stored at -80°C until use. Isolation of microvesicle RNA Isolation of RNA from microvesicles was performed as previously described [18] with a few modifications. Briefly, 1 mL of serum was transferred to an ultracentri- fuge tube, diluted 1:3 with cold PBS and centrifuged at 120,000 g for 80 min at 8°C and the supernatant was carefully aspirated off without disturbing the microvesi- cle pellet. The pellet was resuspended, treated for 15 min with 4 U of DNase I (Ambion, Austin, TX, USA) (in 25 μL of the accompanying buffer), 700 μL miR- Neasy lysis buffer (Qiazol Reagent) (Qiagen, Valencia, CA, USA) was then added to the tube and the RNA was isolated following the manufacturer’s recommendations. After elution of the RNA from the column in 30 μL nuclease-free water (Ambion), the RNA was precipitated by adding 2.5 volumes 100% EtOH, 1/10 3 M sodium acetate (pH 5.2) and incubated at -20°C for 1 h. Samples were then centrifuged for 20 min at 16,000 g and the supernatant was removed. The pellet was left to dry at r.t. and dissolved in 14 μL nuclease-free water and stored at -80°C until needed. RNA quality and concen- tration was assessed with the Agilent Bioanalyzer RNA Pico Chip and the Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). Linear amplification and array hybridization Linear amplification and hybridization to Agilent micro- arrays was carried out by Miltenyi, according to manu- facturer’s recommendations. Briefly, exoRNA was linearly amplified and fluorescently labeled with Cy3 using Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA, USA) and 1.4 μg amplified RNA was hybridized to Agilent 4×44K Human Microarrays, washed and scanned. Raw data was generated by image analysis using Feature Extraction (Agilent). Microarray data analysis The raw data exported from Agilent Feature Extraction v9.1 was pre-processed and normalized using R/