|
| Status |
Public on Aug 21, 2016 |
| Title |
pp3_10mcM_8h_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: 8h
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 (3) |
| GSE50145 |
Proteasome inhibition and oxidative biochemistry are synergistic triggers of metabolic heme toxicity (part 1) |
| GSE50146 |
Proteasome inhibition and oxidative biochemistry are synergistic triggers of metabolic heme toxicity (part 2) |
| GSE50147 |
Proteasome inhibition and oxidative biochemistry are synergistic triggers of metabolic heme toxicity |
|
