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Small-sample protocol recommendations, and GeneChip hybridization followed our standard protocol [28]. After washes, arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). Light intensity data from all spots on each chip were recorded as .cel files, stored on an internal server, and written to a digital variable disc (DVD).Data normalization and filteringreproducibility
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Cal significance.Paraffin sections of our patient-derived glioblastoma xenografts (15 of 22 lines) were stained for galectin-1 expression. Around half of the xenografts tested showed preferential staining at the tumor-brain interface (Figure 3). A few tumors stained in their entirety, and another subset lacked significant staining. The 2 to 4 fold change in galectin-1 mRNA expression at the tumor
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Ed to their corresponding extraction buffer reservoirs. The column-based PicoPure RNA isolation kit (Arcturus, Mountain View, CA) was used to extract RNA from collected cells per manufacturer instructions.Toussaint et al. Molecular Cancer 2012, 11:32 http://www.molecular-cancer.com/content/11/1/Page 3 ofFigure 1 Three geographic tumor regions targeted for RNA isolation. Using laser-capture microdi
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Assays of GBM cells stably transfected to over-express galectin-1, perfectly fit in with the previous studies mentioned above and highlight the importance of galectin-1 in the biologically aggressive behavior of experimental GBMs. While there was no enhancement of proliferation or change inattachment to fibronectin, galectin-1 upregulation induced more rapid two-dimensional migration and enhanced
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Normal brain) and totalRNA was eluted at the final step into a final volume of 11 microliters. One microliter of each eluted RNA sample was used for quantitation with the RiboGreen (Molecular Probes, Eugene, OR) assay kit. These total RNA samples were analyzed for integrity by obtaining electropherograms on an Agilent 2100 Bioanalyzer chip. Samples of acceptable quality based on RNA integrity numb
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Galectin-1 transfectants. A population of GFP-sorted cells (the "Gal-1" bars in Figure 4A) was compared to its parental counterpart. The number of metabolically-active cells attached to fibronectin was no different between the two lines at eight hours. Changing the media at four hours reduced the number of cells left for labeling, but the effect was equal in both groups, suggesting a similar rate
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Of each cell colony over 24 hours was calculated. Data for each cell line was averaged over 10 wells and compared between parental, control transfected, and galectin-1 transfected cells. A t-test was applied to compare means.In vitro invasion assay6.8 pg +/- 4.2 pg. In spite of this variability, the quality of the RNA was consistently high with a mean RNA integrity number of 8.13 ?0.74. Expression
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That is relatively over-expressed at the tumor periphery. These graphical representations of gene expression data compare the relative expression of galectin-1 from the core and edge of tumors to pooled data from normal mouse brain samples. (Graphics from GeneSpringW).created. To ensure that galectin-1 over-expression would not enhance proliferation of the U87MG line (and hence alter the interpret