Additional file 3: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Figure S2. A longitudinal study on a single GBM patient was carried out in order to monitor the changes in serum HOTAIR expression over time. 3 different time points were included in this study: pre-op (the blood was drawn right before the surgery started), post-op (at least 24 h after surgery) and during the 2 week follow-up (F/U) with the neurosurgeon. We show that the level of HOTAIR decreases after surgery and at the follow-up visit. (PDF 430 kb

Additional file 1: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Supplementary materials and methods. (DOCX 109 kb

Additional file 2: of Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Figure S1. HOTAIR expression detected in our biomarker assay is derived mostly from circulating RNA, not DNA. 3 GBM serum samples were selected at random and the relative HOTAIR expression in GBM serum with and without reverse transcription (RT)-PCR was determined. The HOTAIR RNA was reverse-transcribed into HOTAIR cDNA and qPCR was performed. The circulating HOTAIR DNA in the serum was detected by qPCR without RT. The considerable difference between HOTAIR expression with and without RT demonstrates that the HOTAIR we are detecting in our qRT-PCR reactions is derived from RNA and not DNA. (PDF 1003 kb

Gene Networks created by Pairs with high PCC (greater than 0.7) and high hypergeometric p-value yield less experimentally verified interactions.

<p>The number of connections identified was calculated for gene pairs with high PCC and high hypergeometric p-values. These connections were then compared to those identified in the literature. Note that few connections were found to be experimentally validated.</p

Pipeline for identifying patient-specific gene association in GBM.

<p>Our first step in our pipeline is to identify Differentially Expressed (DE) genes that are represented in 3 out of 4 algorithms. Next, we filter this DE gene list for those genes that overlapped with DE genes in the TCGA GBM Database. We then calculate the Correlation Coefficient and a hypergeometric p-value for every gene pair. Finally, by selecting the gene pairs with the highest correlation values we create a patient specific gene correlation network, which can be experimentally verified. As a starting point for our experiments, we can use the sub-networks in which, already verified connections exist in the literature.</p

Correlation networks created by using the top gene pairs for each patient.

<p>The number of connections we identified were compared to those previously described in the literature (red). Yellow indicates connections, which were identified in protein-protein interaction databases.</p

Figure 2

<p>A. Recostrution of the distribution of NCs superimposed on a coronal section immunoreacted with anti-MAP-2 antibody (immunoperoxydase staining) to identify the ischemic region. Green fluorescent CFDA-stained stem cells were counted and plotted on the adjacent section stained with the MAP-2 fluorescent antibody (calibration bar = 1 cm). In B and C, microphotographs of green fluorescent CFDA-stained stem cells on sections counterstained with the fluorescent anti-MAP-2 antibody are shown at ×10 (B) and ×40 (C) magnifications. The pictures were taken in the transition region between the MAP-2⁺ and the MAP-2⁻ areas in the olfactory region. Calibration bars: 150 µm and 16 µm for B and C, respectively.</p

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Figure 4

<p>(A) Changes in extracellular pH <u>(pH_e)</u> in the PC and in mOT induced by ipsilateral MCA occlusion and reperfusion. Occlusion induced a rapid metabolic acidification of the extracellular microenviroment in PC, interrupted by a transient and mild basification (arrow) associated to the hypoxic spreading depression (HD, asterisk). No changes in extracellular [H⁺] were recorded in the mOT, that is not served by the MCA. (B) Simultaneous changes in extracellular potassium concentration ([K⁺]_o)and extracellular pH in the PC after MCA occlusion and reperfusion. An initial enhancement in [K⁺] was followed by a fast and large increase in [K⁺]_o. associated to the HD. The schematic drawing on the right illustrates the position of the two-barrel recording electrodes. The field responses (FP) recorded with the conventional extracellular barrel are also shown. The period of MCA occlusion is marked by the shaded area.</p

Mean NCs density (cells/cm²) in the MAP-2 positive zone contralateral (left column) and ipsilateral (middle column) to the ischemic side and in the MAP-2 negative zone of the ischemic hemisphere (right column) in 6 different experiments.

<p>On the left, the number of slices on which counts were made for each experiments is indicated.</p