|
| Status |
Public on Apr 25, 2014 |
| Title |
U87-EV human glioblastoma xenograft - Control - 3 [HG-U133_Plus_2] |
| Sample type |
RNA |
| |
|
| Source name |
Control
|
| Organism |
Homo sapiens |
| Characteristics |
specific-host: BALB/c SCID mouse
tissue type: U87-EV human glioblastoma xenograft tumour
|
| Treatment protocol |
When tumors reached the size of 0.35-0.45 cm3, the mice were treated by intraperitoneal injection (i.p.) of Bevacizumab (10mg/kg body weight), dibenzazepine (DBZ; Syncom Groningen, The Netherlands) (8.1mmol/kg) (Li et al., 2011) or phosphate buffered saline (PBS) as a control. After 3 days, a second dose of Bevacizumab or DBZ was given and, 4h later, the mice were sacrificed and the tumors were collected. Half of each tumor sample was freshly frozen in liquid nitrogen and subsequently used for RNA extraction.
|
| Growth protocol |
All protocols were carried out under Indiana University Institutional Animal Care and Use Committee (IACUC), and UK Home Office approved protocols and regulations. 107 Human glioblastoma U87GM cells were were subcutaneously implanted into 7 to 8-week-old female BALB/c SCID mice (Harlan Sprague Dawley, Inc., Indiana), 100μl of cell suspension mixed with an equal volume of matrigel (BD Bioscience). Each group consisted of 5 mice. Tumor growth was measured using a caliper and calculated from the formula: “V = L x W x H x π / 0.52”.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Xenograft samples were placed in RNAlater (Ambion, USA) overnight before storage at -80oC. RNA was extracted using Tri-reagant (Sigma-Aldrich) and washed using the Qiagen Spin column (Qiagen). RNA quality and quantity were confirmed using the NanoDrop ND-1000 spectrophotometer and the Agilent 2100 bioanalyzer (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturer’s recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in ~100-fold amplification of RNA. A complete description of procedures is available at: http://bioinf.picr.man.ac.uk/mbcf/Downloads/GeneChip_Target_Prep_Protocol-CR-UK_v4.pdf
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| |
|
| Hybridization protocol |
Target cDNA generated from each sample was then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, spike controls were added to 10 µg fragmented cRNA before overnight hybridization to HGU133plus2 arrays. Arrays were then washed and stained with streptavidin-phycoerythrin, before imaging on an Affymetrix GeneChip (3000) scanner. A complete description of these procedures is available at http://bioinf.picr.man.ac.uk/mbcf/Downloads/GeneChip_Hyb_Wash_Scan_Protocol-CR-UK_v4.pdf.
|
| Scan protocol |
Affymetrix GeneChip (3000) scanner
|
| Description |
tumor tissue includes mouse host stroma cells
|
| Data processing |
Following scanning, .DAT files were processed using Affymetrix GCOS, 1.1.1.052, to generate .CEL files. Subsequent data processing was performed on these data using the affy, simpleaffy and GCRMA packages from BioConductor and R. mRNA was estimated using GCRMA and log2 values were used.
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| |
|
| Submission date |
Jul 10, 2012 |
| Last update date |
Apr 25, 2014 |
| Contact name |
Francesca Buffa |
| E-mail(s) |
francesca.buffa@imm.ox.ac.uk
|
| Organization name |
University of Oxford
|
| Street address |
The Weatherall Institute of Molecular Medicine
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE39223 |
Regulation of gene expressions in vivo by anti-VEGF and anti-Notch therapy [HG-U133_Plus_2] |
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