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On began the day after transfection. When enough GFP-expressing cells were identified, single cell suspensions were sorted under sterile conditions using the GFP filters (488/530 nm excitation/emission) on a FACS Vantage Sorter (Beckton Dickinson Immunocytometry Systems, San Jose, CA). Cells were collected in 96-well plates at a setting of 2 cells / well, after attempts to collect 1 cell per well
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S independently with similar results. Statistical significance was determined by one-way ANOVA followed by Bonferroni's post hoc analysis for multiple comparisons. *Significantly different compared with PBStreated control (P
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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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By the ample amount of normal mouse brain tissue available for dissection. In spite of species differences, cross-hybridization of mouse genetic material to human probes did prove to be a common occurrence. These data made it possible to control, rather stringently, for the potential contamination of tumor edge samples with mouse brain. Of course, there could still be possible contamination ?react
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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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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
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Few of our U87 galectin1 clones. Parental U87MG cells, along with galectin-1 and acGFP-only clones were injected into the right caudate/putamen complex of nude mice. Tumors overexpressing galectin-1 shortened survival of their hosts compared to their parental counterparts (Figure 5). A few animals (7/20) bearing tumors expressing acGFP alone eventually exhibited neurological symptoms. The examinat