|
| Status |
Public on Feb 25, 2013 |
| Title |
shRNA-control-rep-2 |
| Sample type |
RNA |
| |
|
| Source name |
A375P cells expressing Control shScr
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Melanoma cell line
cell line name: A375P
expression: Control shScr
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy (Qiagen) according to the manufacturer's instructions. The Dana Farber Cancer Institute Microarray Core synthesized single-stranded cDNA from the extracted RNA. RNA ws 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.
|
| Label |
biotin
|
| Label protocol |
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. The biotinylated complementary RNA (cRNA) waas purified from the IVT reaction mixture using magnetic particles. The cRNA was quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. The purified cRNA was fragmented in order to facilitate the subsequent hybridization step.
|
| |
|
| Hybridization protocol |
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 hybridization solution was then removed. The chips were transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA was 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 was excited using a confocal laser scanner, GCS3000, and the positions and intensities of the fluorescent emissions were captured.
|
| Data processing |
The data were analyzed with RMA (Gene Pattern's ExpressionFileCreator version 8). Parameters: method RMA quantile normalization yes background correct yes compute present absent calls no normalization method none value to scale to clm file annotate probes yes The data were analyzed with RMA (Gene Pattern's ExpressionFileCreator version 8). Parameters: method RMA quantile normalization yes background correct yes compute present absent calls no normalization method none value to scale to clm file annotate probes yes.
|
| |
|
| Submission date |
Mar 28, 2012 |
| Last update date |
Feb 25, 2013 |
| Contact name |
Pere Puigserver |
| E-mail(s) |
pere_puigserver@dfci.harvard.edu
|
| Phone |
617-582-7977
|
| Organization name |
Dana-Farber Cancer Institute
|
| Street address |
450 Brookline Av. CLSB-11144
|
| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE36879 |
Profiling A375P melanoma cells following PGC1a suppression |
|
