|
| Status |
Public on Nov 11, 2008 |
| Title |
6_noTx |
| Sample type |
RNA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
Multiple Myeloma MM.1S cells, not treated, time 6 hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from enzastaurin-treated or untreated MM.1S cells using TRIzol reagent (Invitrogen, Carlsbad, CA).
|
| Label |
biotin
|
| Label protocol |
Synthesis of double-stranded cDNA which then serves as a template for the synthesis of biotinylated cRNA by in vitro transcription. RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out. The double-stranded cDNA is 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. The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample. The purified cRNA is fragmented in order to facilitate the subsequent hybridization step.
|
| |
|
| Hybridization protocol |
The fragmented cRNA is 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 are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner (Gene Array Scanner (Affymetrix)), and the positions and intensities of the fluorescent emissions are captured.
|
| Description |
none
|
| Data processing |
CEL files were obtained using the Affymetrix Microarray Suite 5.0 software. 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 |
Nov 07, 2008 |
| Last update date |
Nov 11, 2008 |
| Contact name |
Marc Raab |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
Dana Farber Cancer Institute
|
| Street address |
44 Binney Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE13514 |
Targeting PKC: A Novel Role for beta-catenin in ER stress and Apoptotic Signaling |
|
