|
| Status |
Public on Oct 30, 2011 |
| Title |
LSK WT CONTROL REP2 |
| Sample type |
RNA |
| |
|
| Source name |
LSK WT CONTROL
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57Bl/6J
genotype/variation: control wild type mice
cell type: LSK cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA fraction. RNA Integrity Numbers were in the range 9.1 to 10.0 when assayed by Lab-chip technology on an Agilent 2100 Bioanalyzer. Available RNA amounts in the range of 3-27 ng were subjected to a preliminary amplification step with a TransPlex Whole Transcriptome Amplification WTA2 kit (Sigma-Aldrich). Optimal cycle number was set at 17, by proceeding 2–3 cycles beyond the amplification “plateau” as observed with real-time qPCR.
|
| Label |
Cy3
|
| Label protocol |
Amount of nucleic acid labeled: 250ng. Commercial Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis. ULS Labeling for Blood, Cells, Tissues or FFPE (with a High Throughput option) version 3.2 kit was used. Recommendations from Sigma-Aldrich for the integration of TransPlex WTA with the Agilent microarray workflow were followed, such as the omission of Cot-1 DNA. Briefly, cDNA is non-enzymatically labeled with ULS technology (Kreatech), in which a platinum dye complexed with Cy3 fluorophore couples preferentially with guanine molecules of the sample. Labeled samples are purified with gel filtration columns.
|
| |
|
| Hybridization protocol |
Hybridization chamber type: SureHyb hybridization chamber (Agilent). Quantity of labeled extract used: ~190-250 ng. Volume: 40 uL, Temperature: 65C, Duration: 40 hours.
|
| Scan protocol |
Scanned on an G2505C DNA microarray scanner (Agilent). Images were quantified using Agilent Feature Extraction Software (ver. 10.7).
|
| Description |
VBB71WT
|
| Data processing |
Hybridized microarray images were analyzed and backgound subtracted with Agilent FeatureExtraction (FE) Software. Raw data was subsequently normalized with GeneSpring software. Threshold raw signals to 1, Log base2 transformation, Global intra-array normalization (normalization of the distribution of the overall expression level of all genes for each sample, which is assumed to be constant): Shift to 75.0 percentile, Baseline Transformation: the value per-gene in each sample is centered by subtracting the median for that same gene in all samples. The term ''raw'' signal values refer to the linear data after thresholding and summarization. ''Normalization'' comprises log transformation, intra-array quantile normalization (to 75th percentile of all measurements from the same array) and per-gene transformation (signal from each probe in the array to the median of the corresponding signals across all arrays). [More details on the analysis are provided as Series supplementary files: analysis.pdf & analysis_result.xls]
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| |
|
| Submission date |
Aug 16, 2011 |
| Last update date |
Oct 30, 2011 |
| Contact name |
Lorena Arranz |
| E-mail(s) |
larranz@cnic.es
|
| Organization name |
CNIC
|
| Department |
CARDIOVASCULAR DEVELOPMENT AND REPAIR
|
| Lab |
Stem Cell Aging
|
| Street address |
MELCHOR FERNANDEZ ALMAGRO
|
| City |
MADRID |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE31404 |
Expression profiling of LSK cells from Bmi1 cKO mice in hematopoiesis versus LSK control cells from WT mice |
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