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Sample GSM703373 Query DataSets for GSM703373
Status Public on Oct 05, 2011
Title MOUSE4_2.0mm_UV
Sample type RNA
 
Channel 1
Source name Dorsal skin, UV treated
Organism Mus musculus
Characteristics mouse identifier: 4
strain: SKH1
age: 3-4 months
tissue: dorsal skin
biopsy punch diameter: 2.0mm
treatment: UVB radiation
Treatment protocol Two mice were untreated controls and two mice were irradiated with 300 J/m2 UVB light. 24 hours after exposure, both treated and untreated mice were euthanized by cervical dislocation. This was followed directly by biopsy sampling from the back to prevent RNA decay. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80 °C until further processing.
Growth protocol Four 3- to 4-month-old SKH1 hairless mice were used in this experiment.
Extracted molecule total RNA
Extraction protocol Biopsy samples were grinded individually with a liquid nitrogen pre-chilled metal micro-pestle (Carl Roth) to a fine powder. The powder was transferred to a 1.5ml tube containing 300 µl Qiazol (Qiagen). The homogenate was vortexed vigorously for 15 s, shaken for 10 min on a Heidolph REAX 2000 with a multi-test tube attachment (speed setting 6), and then spun down quickly for 15 s. 60 µl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred as a whole to a pre-prepared phase-lock gel heavy containing tube. The tubes were inverted and centrifuged for 15 min at 12,000xg. The aqueous phase was transferred to a new 1.5 ml tube. The RNA was purified by column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) – Appendix D: RNA Cleanup after Lysis and Homogenization with Qiazol Lysis Reagent (Qiagen). At the end of the procedure, the RNA was eluted in 14 µl nuclease-free water.
Label Cy3
Label protocol Per RNA sample, 200 ng total RNA was amplified according to the Agilent LRILAK kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis (2.5 mM rGAU (GE Healthcare), 0.75 mM rCTP (GE Healthcare), 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test aRNA samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from the Test samples) was labeled with Cy5. 5 µg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 µl DMSO. To each sample, 10 µl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1h. Reactions were quenched with the addition of 5 µl 4M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.
 
Channel 2
Source name Pooled common reference
Organism Mus musculus
Characteristics composition: pool from all samples
strain: SKH1
age: 3-4 months
tissue: dorsal skin
sample type: reference
Treatment protocol Two mice were untreated controls and two mice were irradiated with 300 J/m2 UVB light. 24 hours after exposure, both treated and untreated mice were euthanized by cervical dislocation. This was followed directly by biopsy sampling from the back to prevent RNA decay. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80 °C until further processing.
Growth protocol Four 3- to 4-month-old SKH1 hairless mice were used in this experiment.
Extracted molecule total RNA
Extraction protocol Biopsy samples were grinded individually with a liquid nitrogen pre-chilled metal micro-pestle (Carl Roth) to a fine powder. The powder was transferred to a 1.5ml tube containing 300 µl Qiazol (Qiagen). The homogenate was vortexed vigorously for 15 s, shaken for 10 min on a Heidolph REAX 2000 with a multi-test tube attachment (speed setting 6), and then spun down quickly for 15 s. 60 µl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred as a whole to a pre-prepared phase-lock gel heavy containing tube. The tubes were inverted and centrifuged for 15 min at 12,000xg. The aqueous phase was transferred to a new 1.5 ml tube. The RNA was purified by column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) – Appendix D: RNA Cleanup after Lysis and Homogenization with Qiazol Lysis Reagent (Qiagen). At the end of the procedure, the RNA was eluted in 14 µl nuclease-free water.
Label Cy5
Label protocol Per RNA sample, 200 ng total RNA was amplified according to the Agilent LRILAK kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis (2.5 mM rGAU (GE Healthcare), 0.75 mM rCTP (GE Healthcare), 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test aRNA samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from the Test samples) was labeled with Cy5. 5 µg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 µl DMSO. To each sample, 10 µl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1h. Reactions were quenched with the addition of 5 µl 4M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.
 
 
Hybridization protocol Each hybridization mixture was made up from 1.1 µg Test (Cy3) and 1.1 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 (Roche 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 the microarrays, and hybridized for 18 hours at 42°C with the Roche NimbleGen Hybridization System 4. Afterwards, the slides were washed according to the Roche NimbleGen Arrays User’s Guide – Gene Expression Arrays Version 5.0.
Scan protocol Arrays were scanned in an ozone-free room with a DNA microarray scanner G2565CA (Agilent Technologies), and feature extraction was performed with NimbleScan v2.5 (Roche NimbleGen).
Description Dual color Nimblegen data.
Data processing No processed data was generated.

In the raw *.FTR file:
SIGNAL_MEAN_433844A**_#1 column represents the Cy3 channel
SIGNAL_MEAN_433844A**_#2 column represents the Cy5 channel
 
Submission date Apr 07, 2011
Last update date Oct 05, 2011
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)
GSE28463 RNA isolation from mouse skin biopies

Supplementary file Size Download File type/resource
GSM703373.ftr.gz 3.9 Mb (ftp)(http) FTR
Processed data not applicable for this record

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