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Sample GSM1243095 Query DataSets for GSM1243095
Status Public on Sep 01, 2014
Title Mm_mouse_4_t_1
Sample type RNA
 
Source name Mm_mouse_4_t_1
Organism Mus musculus
Characteristics time (hours): 1
tissue: skin
genotype: Wild-type (WT; p53+/+)
Treatment protocol All mouse treatments have been approved by the institute's Experimental Animal Ethical Committee and carried out in accordance with national legislation. Mice were UVB irradiated at different doses (90, 180, 360, 450, 720 J/m2) in a chamber containing Phillips TL12 lamps. Control mice were mock treated.
Growth protocol Wild-type (WT; p53+/+) mice were used for UV exposure studies. All mice used were males of 7-10 weeks of age and at least 10 times backcrossed in SKH hairless strain. Mice received normal feed and water ad libitum.
Extracted molecule total RNA
Extraction protocol At different time points after UV-B exposure, both treated and untreated mice were anaesthetized by isoflurane. 1.5mm biopsies were sampled from the back. by punching a half moon shape on folded skin. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA). RNA was assessed for quality with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label Cy3
Label protocol Per test sample, 5 µg total RNA combined with 1 µg random octamers (Biolegio) in 4.5 µl was heated to 65°C for 10 min to denature the RNA and was allowed to cool in an ice-waterbath for 10 min. This 4.5 µl was made to 10 µl with a first strand mastermix containing final concentrations of 50 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 75 mM KCl, 200 mM Raffinose (Sigma-Aldrich), 0.015% Triton X-100, 30 ng Actinomycin-D (Sigma-Aldrich), 0.01M DTT, 0.5 mM dGAC, 0.35 mM dUTP, 0.15 mM dUTP-Cy3 (test) or dUTP-Cy5 (common reference) (GE Healthcare) and 200U SuperScript-II (Life Technologies). This mixture was incubated for 2 min at 25°C, 120 min at 42°C and 15 min at 70°C. Finally, 1.5 µl of 2.5M NaOH was added to hydrolyze the remaining RNA by heating for 10 min at 70°C. 8.5 µl 2M MOPS was added for neutralization and the labeled cDNA was purified with the E.Z.N.A. MicroElute RNA Clean-up Kit (Omega Biotek). Dye incorporation and cDNA yield was measured on the NanoDrop ND-1000 (Thermo Scientific) yielding 2-2.5 µg per sample and a FOI > 10 pmol/µg. The common reference was made by an equimolar pool of all test samples (5 µg per sample) and subsequently labeled as the test samples with Cy5 incorporation.
 
Hybridization protocol Each hybridization mixture was made up from 750 ng Test (Cy3) and 750 µg Reference (Cy5) sample. Samples were dried and 1.98 µl of water was added. The hybridization cocktail was made according to the manufacturer’s instructions (Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0, Roche Nimblegen). 5.22 µl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto 12x135k custom designed microarrays against E. coli (OID 38205, Design 120315). Microarrays were hybridized for 18 hours at 42°C with the NimbleGen Hybridization System 4 (Roche Nimblegen).
Scan protocol the slides were washed according to the Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0 and scanned in an ozone-free room with a Agilent DNA microarray scanner G2565CA (Agilent Technologies). Feature extraction was performed with NimbleScan v2.6 (Roche Nimblegen).
Data processing The raw data from all arrays was subjected to multiple quality control checks, i.e. visual inspection of the scans, testing against criteria for foreground and background signals, testing for consistent performance of the labeling dyes, checking for spatial effects through pseudo-color plots, and inspection of pre- and post-normalized data with box plots, ratio-intensity (RI) plots and PCA plots. Within array normalization was performed using LOESS. Between array normalization was run on the Cy3-channel by summarizing (Genebank id's) the intensity values of the probes in a probe set using the robust multi-array average (RMA) algorithm.
 
Submission date Oct 17, 2013
Last update date Sep 01, 2014
Contact name Oskar Bruning
Organization name University of Amsterdam
Department SILS
Lab MicroArray Department
Street address Science Park 904
City Amsterdam
ZIP/Postal code 1098 XH
Country Netherlands
 
Platform ID GPL10192
Series (1)
GSE51347 Effect of multiple longitudinal biopsy sampling in individual mice

Data table header descriptions
ID_REF
VALUE RMA signal

Data table
ID_REF VALUE
AB000096 10.29891614
AB000490 8.169687596
AB001425 7.829101098
AB001435 8.671561301
AB001539 8.032925886
AB001750 8.945790672
AB001926 11.60334453
AB003502 12.41400297
AB004048 8.656026954
AB005662 7.741222591
AB005665 5.316811075
AB005909 4.705817136
AB006034 6.654870041
AB006103 6.050951706
AB007407 5.907413827
AB008928 12.86114615
AB009369 9.190145914
AB010088 7.278323551
AB010122 10.32113932
AB011499 5.778325783

Total number of rows: 44170

Table truncated, full table size 918 Kbytes.




Supplementary file Size Download File type/resource
GSM1243095_510653A02.ftr.gz 3.3 Mb (ftp)(http) FTR
Processed data included within Sample table

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