|
| Status |
Public on Apr 09, 2007 |
| Title |
XBP1s B cells 4 |
| Sample type |
RNA |
| |
|
| Source name |
XBP1s_B_cells_spleen
|
| Organism |
Mus musculus |
| Characteristics |
XBP1s_B_cells_spleen_20_weeks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
B220+ splenic B cells were isolated using magnetic microbeads from Miltenyi Biotec. Plasma cells were isolated from human bone marrow mononuclear cells from healthy donors (Cambrex BioScience) using immunomagnetic bead selection with mouse anti-human CD138 monoclonal antibodies as described (Zhan, et al., 2002), except that we used LS MACS separation columns (Miltenyi Biotec) and before incubating the bone marrow mononuclear cells to CD138-coated magnetic beads, we removed contaminating histiocytes and macrophages by preloading and washing out the cells in the magnetic columns and field. Total RNA was isolated from control and transgenic B220+ splenic mononuclear cells or myeloma tumors using TRIzol Reagent.
|
| Label |
biotin
|
| Label protocol |
RNA was converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis was then carried out.
The double-stranded cDNA was used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
|
| |
|
| Hybridization protocol |
The biotinylated complementary RNA (cRNA) was purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA was quantified spectrophotometrically and purity of the cRNA was also assessed by spectrophometric measurements.
The purified cRNA was fragmented in order to facilitate the subsequent hybridization step. The cRNA was purified from the fragmentation reaction using phenol/chloroform extraction and ethanol precipitation.
The fragmented cRNA was added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
The chips were then transferred to a fluidics instrument that performed washes to remove cRNA that had not hybridized to its complementary oligonucleotide probe. The bound cRNA was then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors were then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide was excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions were captured. These measures provided the basis of subsequent biostatistical analysis.
|
| Description |
NA
|
| Data processing |
The DNA Chip Analyzer (dChip) was used to normalize all CEL files to a baseline array with overall median intensity, and the model-based expression (perfect match minus mismatch) was used to compute the expression values.
|
| |
|
| Submission date |
Feb 07, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Ronald A DePinho |
| E-mail(s) |
[email protected], [email protected], [email protected]
|
| Organization name |
Dana Farber Cancer Institute
|
| Street address |
44 Binney St. M417
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE6980 |
The Differentiation and Stress Response Factor, XBP-1, Drives Multiple Myeloma Pathogenesis |
|
| Relations |
| Reanalyzed by |
GSE119085 |
