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Sample GSM1214713 Query DataSets for GSM1214713
Status Public on Aug 21, 2016
Title pp3_10mcM_1h_a
Sample type RNA
 
Channel 1
Source name pp3
Organism Mus musculus
Characteristics hmox: positive
treatment: no treatment
incubation time: 0h
cell type: Wild type Mouse embryonal fibroblasts (MEF) (=pp3)
genotype: Wild type
Treatment protocol na
Growth protocol na
Extracted molecule total RNA
Extraction protocol After washing and lysing the cells with Buffer RLT, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions, including an on-column DNA digestion step (RNase-Free DNase Set; QIAGEN). To ensure that only high quality RNA (RIN >7.0) was used for gene expression analysis, each RNA sample was checked on a RNA Nanochip with a Bioanalyzer 2100 (Agilent Technologies, Basel, Switzerland). RNA was quantified spectrophotometrically with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Label Cy3
Label protocol Fluorescently labeled cRNA was generated from 500 ng total RNA with the Quick Amp Labeling Kit (Agilent) according to Agilent’s Two-Color Microarray-Based Gene Expression Analysis Protocol Version 5.7, March 2008 (Agilent publication number G4140-90050). In short, total RNA was reverse transcripted to cDNA using T7 Promotor Primer and MMLV-RT. Then cDNA was converted to cRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
 
Channel 2
Source name pp3
Organism Mus musculus
Characteristics hmox: positive
treatment: 10mcM heme
incubation time: 1h
cell type: Wild type Mouse embryonal fibroblasts (MEF) (=pp3)
genotype: Wild type
Treatment protocol na
Growth protocol na
Extracted molecule total RNA
Extraction protocol After washing and lysing the cells with Buffer RLT, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions, including an on-column DNA digestion step (RNase-Free DNase Set; QIAGEN). To ensure that only high quality RNA (RIN >7.0) was used for gene expression analysis, each RNA sample was checked on a RNA Nanochip with a Bioanalyzer 2100 (Agilent Technologies, Basel, Switzerland). RNA was quantified spectrophotometrically with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Label Cy5
Label protocol Fluorescently labeled cRNA was generated from 500 ng total RNA with the Quick Amp Labeling Kit (Agilent) according to Agilent’s Two-Color Microarray-Based Gene Expression Analysis Protocol Version 5.7, March 2008 (Agilent publication number G4140-90050). In short, total RNA was reverse transcripted to cDNA using T7 Promotor Primer and MMLV-RT. Then cDNA was converted to cRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
 
 
Hybridization protocol After purification of amplified cRNA according to the QIAgen RNeasy purification protocol (QIAGEN) 825 ng of Cy3 and Cy5 labeled cRNA were competitively hybridized for 17 hours at 65° C in a hybridization oven (Agilent G2545A) set to 10 rpm in GEx Hybridization buffer HI-RPM according to the manufacturer's protocol (Agilent’s Two-Color Microarray-Based Gene Expression Analysis). Washing was performed according to the recommended protocol including the Stabilization and Drying Solution step.
Scan protocol Array slides were XDR scanned at 5 mcm resolution on a Agilent Microarray High-Resolution C Scanner (G2505C) with 100% PMT and 10% for lower intensity.
Description Wild type Mouse embryonal fibroblasts (MEF) (=pp3)
Data processing Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software Version 10.7.3.1. Spot values were normalized using the default linear-lowess normalization. To identify differentially expressed features, calculated log10 ratio values (Cy5/Cy3) were imported into JMP Genomics 5.1 without further normalization and the built-in Basic Expression Workflow (ANOVA, LSMeans Differences) was performed.
We don't provide a Matrix Table as we imported the calculated and lowess-normalized log10 ratios of the Feature Extraction result text files directly into JMP Genomis 5.1 and performed the statistical analysis without further normalization.
 
Submission date Aug 23, 2013
Last update date Aug 21, 2016
Contact name Christian Andreas Schaer
Organization name University Hospital Zurich
Street address Raemistrasse 100
City Zurich
ZIP/Postal code 8091
Country Switzerland
 
Platform ID GPL10333
Series (2)
GSE50145 Proteasome inhibition and oxidative biochemistry are synergistic triggers of metabolic heme toxicity (part 1)
GSE50147 Proteasome inhibition and oxidative biochemistry are synergistic triggers of metabolic heme toxicity

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 2.372832125e-002
2 0.000000000e+000
3 0.000000000e+000
4 0.000000000e+000
5 0.000000000e+000
6 0.000000000e+000
7 0.000000000e+000
8 0.000000000e+000
9 0.000000000e+000
10 0.000000000e+000
11 0.000000000e+000
12 2.578305100e-001
13 2.740173336e-001
14 6.229592280e-002
15 2.922371333e-001
16 -2.407499212e-002
17 7.876720494e-002
18 0.000000000e+000
19 0.000000000e+000
20 5.202121543e-002

Total number of rows: 44397

Table truncated, full table size 1002 Kbytes.




Supplementary file Size Download File type/resource
GSM1214713_pp3_10mcM_1h_a.txt.gz 4.2 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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