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Status |
Public on Aug 21, 2020 |
Title |
U87IL33 DNLS c4-4 |
Sample type |
RNA |
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Source name |
Mutant
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Organism |
Mus musculus |
Characteristics |
strain: SCID age: 6-8 weeks tissue: Brain implanted cells: Human Glioma U87 expressing IL-33 DNLS
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Treatment protocol |
U87pcDNA or U87 IL-33 or U87 IL-33 DNLS human glioma cells (5×10^4 cells/mouse) were implanted into the brain of mice by intracranial injection. Mice were monitored weekly. Animals bearing U87 tumors were sacrificed at 1 week after tumor implantation.
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Growth protocol |
Six- to 8-week-old female SCID mice were housed in groups of three to five and maintained on a 12-hr light/dark schedule with a temperature of 22 °C±1 °C and a relative humidity of 50 ± 5%. Food and water were available ad libitum. All procedures were reviewed and approved by the University of Calgary Animal Care Committee. table transfectants of U87 or U251 cells were maintained in complete medium [Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate at 37ºC in a humidified 5% CO2 incubator. Cells were passaged by harvesting with Puck’s EDTA when they reached 80%–90% confluence.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from intracranial xenografts derived from U87pcDNA, IL-33 and DNLS cells. RNA was isolated using mirVana miRNA isolation kit (Ambion) according to the manufacturer's protocol. Total RNA purification to remove genomic DNA was carried out with Qiagen kit of RNeasy® Plus Micro following the manufacturer’s protocols. RNA Integrity Number (RIN) was assessed with RNA 6000 NanoChip on Agilent 2100 Bioanalyzer (Agilent Technologies).
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Label |
Biotin
|
Label protocol |
100 ng of total RNA in a final volume of 5 μl was mixed with a 3′ biotinylated capture probe and a 5′ reporter probe tagged with a fluorescent barcode from the nCounter Inflammation Panel (Mouse v2) gene expression code set.
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Hybridization protocol |
Probes and target transcripts were hybridized overnight at 65°C for 16 hours per the manufacturer’s protocol. Hybridized samples were purified on the NanoString nCounter Prep Station using the high-sensitivity protocol, in which excess capture and reporter probes were removed and transcript-specific ternary complexes were immobilized on a streptavidin-coated cartridge.
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Scan protocol |
The samples were scanned at maximum scan resolution on the nCounter Digital Analyzer.
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Data processing |
Data analysis with nSolver software (NanoString Technologies). Samples that passed QC were further analyzed for fold change and normalized expression levels. The expression levels of each gene were normalized, to both positive control probes and housekeeping genes built-in each probe set, by the nSolver Analysis Software.
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Submission date |
Jun 29, 2020 |
Last update date |
Aug 22, 2020 |
Contact name |
Stephen M Robbins |
E-mail(s) |
srobbins@ucalgary.ca
|
Phone |
(403) 220-4304
|
Organization name |
University of Calgary
|
Department |
Oncology
|
Lab |
HMRB 357
|
Street address |
3330 Hospital Drive N.W.
|
City |
Calgary |
State/province |
Alberta |
ZIP/Postal code |
T2N 4N1 |
Country |
Canada |
|
|
Platform ID |
GPL20885 |
Series (2) |
GSE153488 |
Mouse immune cells response Interleukin-33 (IL-33) [nanostring] |
GSE153489 |
Cerebral immune cells in the Interleukin-33 (IL-33) driven tumor microenvironment |
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