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Sample GSM2892621 Query DataSets for GSM2892621
Status Public on Apr 17, 2018
Title A549res-34µM_2
Sample type RNA
 
Source name NSCLC, cisplatin resistant, 24h cisplatin, 34uM
Organism Homo sapiens
Characteristics sensitivity: resistant
treatment conc: 34
Treatment protocol For all experiments, cells were allowed to attach overnight, experienced 4 h of serum starvation and were subsequently treated with cisplatin for 24 h in IMDM medium without any supplements. The parental cells were treated with 11 µM cisplatin. The resistant sub-line was exposed to 11 µM cisplatin and additionally treated with 34 µM cisplatin. The control cells were treated with the drug-free medium.
Growth protocol The human NSCLC cell line A549 was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Its cisplatin-resistant sub-line A549rCDDP2000 derived from the Resistant Cancer Cell Line (RCCL) collection (www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html) had been established by adapting the growth of A549 cells in the presence of increasing concentrations of cisplatin until a final concentration of 2000 ng/mL cisplatin. A549 cells were grown in IMDM medium (PAN-Biotech, Aidenbach, Germany) containing 4 mM L-glutamine supplemented with 10 % foetal calf serum, 100 I.E./mL penicillin and 0.1 mg/mL streptomycin. The medium of the A549rCDDP2000 cells additionally contained 2 µg/mL cisplatin. Cells were cultivated as monolayers in a humidified atmosphere at 37 °C and 5 % CO2.
Extracted molecule total RNA
Extraction protocol Total ribonucleic acid (RNA) was isolated from the cells with my-Budget RNA Mini Kit (Bio-Budget, Krefeld, Germany) through different spin columns according to the manufacturer’s instructions. Isolated RNA was stored at -80 °C until analysis was performed.
Label Cy3
Label protocol Transcriptome was then analysed using One-Color Whole Genome Array SurePrint G3 Human GE V2 8x60K Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, total RNA was transcribed to complementary deoxyribonucleic acid (cDNA) using AffinityScript-RT, Oligo dT-Promoter Primer and T7 RNA Polymerase and labelled using the One Colour RNA Spike-In Kit (positive controls) including Cyanin 3-CTP (Cy3) dye.
 
Hybridization protocol After purifying the labelled/amplified complementary RNA (cRNA) using silica-membrane RNeasy spin columns from the RNeasy® Mini Kit (Qiagen, Venlo, the Netherlands), cRNA was quantified spectrophotometrically using NanoDropTM ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). 40 µL of equivalent amounts of Cy3-labelled cRNA in 10x Blocking Agent and 25x Fragmentation Buffer, diluted with 2x GEx HI-RPM Hybridization Buffer were loaded on the gaskets of the microarray slide and kept at 65 °C for 17 h with 10 rpm of agitation.
Scan protocol After washing twice with different washing buffers, the microarray was read out with the SureScan Microarray Scanner System (Agilent Technologies, Santa Clara, CA, USA) to obtain immunofluorescence intensity.
Description gene expression in res. cells after treatment with 34µM
Data processing Array data were pre-processed via background correction (exponential convolution method) and quantile normalization. Statistical significances of dose- and resistance-induced gene expression changes were analysed using limma, a linear model-based technique. Differential expression was declared at a 5 % false discovery rate (FDR) cut-off together with an at least twofold up- or downregulation. The overall significance of the signature of differentially expressed genes was assessed via a global test. VALUE contains quantile normalized data for probes with REFSEQ IDs only (29,729)
 
Submission date Dec 18, 2017
Last update date Apr 17, 2018
Contact name Ganna Kalayda
Organization name University of Bonn
Department Clinical Pharmacy
Street address An der Immenburg 4
City Bonn
ZIP/Postal code 53121
Country Germany
 
Platform ID GPL17077
Series (1)
GSE108214 Key players of cisplatin resistance: towards a systems pharmacology approach

Data table header descriptions
ID_REF
VALUE quantile

Data table
ID_REF VALUE
A_23_P117082 13.73645537
A_33_P3246448 5.327100302
A_33_P3318220 5.54423048
A_21_P0000509 18.57168936
A_21_P0000744 11.80900048
A_24_P215804 8.394135892
A_23_P110167 12.43362034
A_33_P3211513 8.436417671
A_23_P103349 5.545026888
A_32_P61480 5.505331886
A_33_P3788124 5.522417501
A_33_P3414202 7.078284292
A_33_P3316686 9.267662012
A_33_P3300975 6.193208247
A_33_P3263061 12.55010165
A_24_P278460 8.729557008
A_21_P0014651 9.578210589
A_23_P340890 10.34103792
A_21_P0010885 12.10593062
A_23_P89762 7.975749375

Total number of rows: 29729

Table truncated, full table size 728 Kbytes.




Supplementary file Size Download File type/resource
GSM2892621_SG11440003_253949429103_S001_GE1_1100_Jul11_2_3.txt.gz 3.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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