|
| Status |
Public on Oct 09, 2021 |
| Title |
I451M pool 29 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C2C12 expressing I451M mutant human desmin
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12 (CRL-1772)
genome modification: pcDNA3 modified with the mouse promoter of desmin, c-myc tag, human desmin gene (with mutation I451M), polyA and puroR/AmpiR genes
number of independent culture pooled: 2
number of independent culture pooled: 2
|
| Growth protocol |
C2C12 were cultured in DMEM supplemented with 10% FCS and 1% penicilin-streptomicin. At 80 % confluency cells were passaged once and cultured for 2 days. They were recovered using a cell scrapper and frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). The RNA extracted from 2 independant cultures were pooled to obtain each analyzed sample (for the control and the different experimental conditions tested).
|
| Label |
Cy5
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies). Control samples are labeled with Cy3 and experimental samples are labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
Pool control
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12 (CRL-1772)
|
| Growth protocol |
C2C12 were cultured in DMEM supplemented with 10% FCS and 1% penicilin-streptomicin. At 80 % confluency cells were passaged once and cultured for 2 days. They were recovered using a cell scrapper and frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). The RNA extracted from 2 independant cultures were pooled to obtain each analyzed sample (for the control and the different experimental conditions tested).
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies). Control samples are labeled with Cy3 and experimental samples are labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole Mouse Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies).
|
| Description |
Comparison of the gene expression profile between Desmin mutant (I451M) expressing line versus Non-modified C2C12 cells. Biological replicate 2.
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (p-values). For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware).
grl*txt files contain the Resolver Software allows the export of a gene list with all normalized Cy5/Cy3-log10 ratios, Cy5/Cy3-fold changes, sequence description, p-values, etc., referred to as gene ratio list (of all genes).
ps*txt files contain putative candidate genes with a fold change >2 and p-value <0.01 are summarized in the pre-selected candidate gene list.
|
| |
|
| Submission date |
Oct 08, 2021 |
| Last update date |
Oct 09, 2021 |
| Contact name |
Onnik Agbulut |
| E-mail(s) |
[email protected]
|
| Organization name |
Sorbonne Université
|
| Street address |
7 quai st-bernard
|
| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE185589 |
C2C12 expressing desmin: WT vs Mutations R406W and I451M |
|
