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| Status |
Public on Aug 04, 2023 |
| Title |
#993_IR 2Gy_24 hrs_rep2 |
| Sample type |
RNA |
| |
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| Source name |
human glioblastoma stem cells, 24 hrs after 2Gy radiation
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line established from glioblastoma WHO grade IV
genotype: wild type TP53
cell type: human glioblastoma stem cells
treatment: 24 hrs after 2Gy radiation
|
| Treatment protocol |
Single cell suspensions were prepared by trituration one day before treatment 30 µM ClQ solved in PBS or radiation with 2Gy of IR. Control samples were processed in parallel using the same procedure except the treatment. 24 hrs after the treatment ClQ-treated, IR-treated or untreated control cells were collected by centrifugation, washed twice with PBS, pelleted and snap-frozen in liquid nitrogen. Frozen cell pellets were kept at -80°C until RNA extraction and hybridization on Affymetrix microarrays.
|
| Growth protocol |
Glioblastoma stem cell lines were maintained in serum-free NeuroBasal Complete medium supplemented with B27 component and growth factors bFGF and EGF (10 and 20 ng/mL, respectively).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed using the Trizol (Invitrogen) method according to the manufacturer’s instructions. The samples were treated with DNAse I (Sigma-Aldrich, St. Louis, MO USA). RNA quality was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) microfluidic electrophoresis and only samples with comparable RNA integrity numbers were processed for microarray analysis.
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized using 5 μg of total RNA. The synthesis of the double-stranded cDNA was performed using the WT Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, USA) and the cleanup was done using the GeneChip® Sample Cleanup module (Affymetrix). In vitro transcription was conducted using the WT Target Labeling Kit (Affymetrix). The total amount of the reaction product was purified using the GeneChip® cRNA Sample Cleanup Module (Affymetrix) and quantified with the NanoDrop ND-1000 (Nanodrop (Thermo), Wilmington, DE USA). cDNA synthesis (ss) was done with the WT Target Labeling Kit (Affymetrix) and the total ssDNA (5.5 μg) was cleaved 35–200 bp fragments enzymatically. The degree of fragmentation and the length distribution of the ssDNA were analyzed by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies). Following fragmentation, a terminal labeling reaction (Biotin) was conducted using the WT Labeling Kit (Affymetrix).
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| |
|
| Hybridization protocol |
Following fragmentation, 0.3 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Data processing |
The data analysis consisted of between-array normalization, probe summary, global clustering and PCA-analysis, fitting the data to a linear model and detection of differential gene expression. To ensure that the intensities had similar distributions across arrays, quantile-normalization was applied to the log2-transformed intensity values as a method for between-array normalization. As for the summary of probes a median polish procedure was chosen. Significant changes in the expression of genes between the groups was analyzed by Empirical Bayes statistics by moderating the standard errors of the estimated values. P-values obtained from the moderated t-statistic were corrected for multiple testing with the Benjamini–Hochberg method. P-value adjustments guarantee a smaller number of false positive findings by controlling the false discovery rate (fdr). For each gene, the null hypothesis suggesting there is no differential expression between degradation levels was rejected when its fdr was lower than 0.05. Samples were assessed in a blinded manner.
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| |
|
| Submission date |
Feb 13, 2023 |
| Last update date |
Aug 04, 2023 |
| Contact name |
Ella L Kim |
| E-mail(s) |
[email protected]
|
| Phone |
06131178211
|
| Organization name |
University Medical Center Mainz
|
| Department |
Neurosurgery
|
| Lab |
Experimental Neurooncology
|
| Street address |
Langenbeckstraße 1
|
| City |
Mainz |
| State/province |
Select an option… |
| ZIP/Postal code |
55131 |
| Country |
Germany |
| |
|
| Platform ID |
GPL27927 |
| Series (1) |
| GSE225191 |
Transcriptomic analysis to identify genes associated with chloroquine-mediated radiosensitization of glioblastoma stem cells |
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