|
| Status |
Public on Jul 10, 2020 |
| Title |
MSCDM_ Control_a_M98 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Murine stem cell derived macrophages, no treatment
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Murine stem cell derived macrophages
treatment: no treatment
incubation time: 0h
|
| 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 (green signal)
|
| 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 |
Murine stem cell derived macrophages, used as a control without treatment
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Murine stem cell derived macrophages
treatment: no treatment
incubation time: 0h
|
| 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 (red signal)
|
| 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 |
Note: For the statistical analysis of our array experiments we imported the log10 ratios of the Feature Extraction result text files into JMP Genomis 5.1. As Feature Extraction Software does already a a linear-lowess normalization, we didn't perform any further normalization. That means that our normalized data are already included in the Agilent Feature Extraction Files, which have been uploaded with this Metadata Worksheet. Any further data processing, i.e. filtering for at least 2-fold over- or underexpression of significant features, we performed after the ANOVA analysis. As we understand the MIAME guidelines it is not necessary to provide a Matrix Table with data output from an ANOVA or Hierarchical Clustering analysis.
|
| 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 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 |
Jul 24, 2017 |
| Last update date |
Jul 10, 2020 |
| 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 (1) |
| GSE101793 |
Revisiting the putative role of heme as a trigger of inflammation |
|
