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On problem with IRES vectors ?inability to transcribe the transcript following the IRES sequence if the first MCS is empty. The new pIRES2-acGFP1acGFP1 vector was used as a control for the pIRES2Gal1-acGFP1 construct. Both vectors were sequenced through their multiple cloning sites to ensure no PCRinduced mutations were present.Transfection and stable clone generationParental, control, and galecti
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Eration, Migration, Invasion, Galectin-Backgound In spite of recent advances in the treatment of patients with glioblastoma, the prognosis for those afflicted remains poor. Even when these tumors harbor a favorable gene methylation profile, the newest standard of care, including temozolomide as a chemotherapeutic [1],* Correspondence: Gtoussaint@medicine.tamhsc.edu 1 The Texas Brain and Spine Inst
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Filtering algorithm. This algorithm was designed to minimize the effect of potential contamination of the edge samples with normal mouse brain cells. Relative expression values for each gene from tumor core, tumor edge, and normal mouse brain samples were compared. Genes of interest were identified that met three criteria: a) low expression at tumor core; b) relatively increased expression at tumo
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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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Filtering algorithm. This algorithm was designed to minimize the effect of potential contamination of the edge samples with normal mouse brain cells. Relative expression values for each gene from tumor core, tumor edge, and normal mouse brain samples were compared. Genes of interest were identified that met three criteria: a) low expression at tumor core; b) relatively increased expression at tumo
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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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Thotopically xenografted into the brains of immunocompromized mice. Invasive cells at the tumor periphery were isolated using laser capture microdissection. The mRNA expression profile of these cells was compared to expression at the tumor core, using normal mouse brain to control for host contamination. Galectin-1, a target identified by screening the resulting data, was stably over-expressed in
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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