|
| Status |
Public on Jul 24, 2010 |
| Title |
Sle2C1 B1a r3 |
| Sample type |
RNA |
| |
|
| Source name |
B1a cells from B6.Sle2c1 mice
|
| Organism |
Mus musculus |
| Characteristics |
cell type: B1a
strain: B6.Sle2c1
|
| Growth protocol |
Gene expression was compared between peritoneal cavity (Pc) B cells (B1a) and splenic B (Bs) cells from B6.Sle2c1 and C57BL/6 (B6) mice at 5 mo of age. CD43- Bs cells were isolated with the Myltenyi B cell kit, leading to a population of > 90% B220hi cells. Pc macrophages were removed by a 2 h culture at 370C in 10% FBS in PBS. Non-adherent Pc cells were first submitted to negative selection with a cocktail of biotinylated anti-Thy1.2 and anti-CD3ε Abs, then to positive selection with biotynylated-anti-CD5 Ab. Cells bound to biotynylated Abs were removed with streptavidin-conjugated magnetic beads (Myltenyi). This protocol led to a 80% B220int CD5+ cell population.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with the Qiagen RNeasy Micro Kit.
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized and labeled with the Ovation Biotin RNA Amplification and Labeling System (NuGEN Technologies, Inc.)
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| |
|
| Hybridization protocol |
hybridization to Affymetrix Mouse Genome 430 2.0 arrays.
|
| Scan protocol |
scanned on an Affymetrix GeneChip Analysis System GC3000 7G.
|
| Description |
exp B1a replicate 3
|
| Data processing |
All data analyses were based on the use of “internal standards” as presented in 1.Dozmorov, I. M., and I. Lefkovits. 2010. Internal standard-based analysis of microarray data.1. Analysis of differential gene expressions. Nucl. Acid Res.
Two-step normalization procedure (1). The first step is the determination of the parameters of the background of the array – average (Av) and standard deviation (SD) using a special iteration procedure. Data in each array are normalized making these parameters equal zero and one correspondingly. After this normalization the gene expressions are presented in the units of the SD of background. We accept S =3 SDs above the mean background level as the preliminary criterion for distinguishing expressed from non-expressed genes. Only genes expressed above background are used for the second step “adjustment”. The second step is the adjustment of the normalized profiles to each other by robust regression analysis of genes expressed above background. This procedure is based on the selection of equally expressed genes as a homogenous family of genes with normally distributed residuals defined as deviations from the regression line. The parameters of this distribution are obtained by the iterative procedure similar to the one used before for the selection of the kernel part of normally distributed background noise. Outliers are thereafter determined as having deviations n associated with this internal standard of equality of expression including thousands members. The functions used for normalization were implemented in Matlab and in Bioconductor package diffGeneAnalysis.
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| |
|
| Submission date |
Jul 23, 2010 |
| Last update date |
Jul 23, 2010 |
| Contact name |
Igor M Dozmorov |
| E-mail(s) |
igor-dozmorov@omrf.org
|
| Phone |
405-271-7052
|
| Fax |
405-271-7063
|
| URL |
http://omrf.org
|
| Organization name |
OMRF
|
| Department |
Arthritis & Immunology
|
| Street address |
825 NE 13th Street
|
| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73162 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE23114 |
Cell cyclin kinase inhibitor Cdkn2c regulates B cell homeostasis and function in the NZM2410-derived murine lupus susceptibility locus Sle2c1 |
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