|
| Status |
Public on Mar 15, 2010 |
| Title |
Control_N-43 [mRNA] |
| Sample type |
RNA |
| |
|
| Source name |
Normal gonadal control replicate 2 of 3
|
| Organism |
Homo sapiens |
| Characteristics |
tumor type: control
tumor composition: N/A
anatomical site: Postpubertal testis
gender: Male
age (yrs): N/A
tumor stage: N/A
|
| Growth protocol |
Cell lines cultured under standard tissue culture conditions - incubated at 37C and in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Total RNA from each sample was used to prepare biotinylated target RNA in a one-cycle target labeling procedure, with minor modifications from the manufacturer’s recommendations available at: http://www.affymetrix.com/support/technical/manual/expression_manual.affx. Briefly, 10 µg of cleaned total RNA was used to generate first-strand cDNA by using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 10-fold amplification of RNA.
|
| |
|
| Hybridization protocol |
Target cRNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx) at Geneservice Ltd (Cambridge Science Park, Cambridge, UK), formerly MRC Geneservice. Briefly, spike controls were added to 15 µg fragmented cRNA before overnight hybridization with the Genechip Human Genome U133A arrays (Affymetrix).
|
| Scan protocol |
Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.
|
| Description |
N/A
|
| Data processing |
The array, once scanned, was used to generate an image file (with the extension .dat). GeneChip Operating Software (GCOS) was then used to compute cell intensity data from the image file and this data was saved in a file with the extension .cel. The .cel (CEL) files were provided by Geneservice Ltd and used for subsequent data analysis. Pre-processing of microarray data (including background correction and normalization) was performed using the Bioconductor package Robust Multi-Array Average (RMA) in the statistical software environment R.
|
| |
|
| Submission date |
Sep 17, 2009 |
| Last update date |
Mar 11, 2010 |
| Contact name |
Matthew Jonathan Murray |
| E-mail(s) |
mjm16@cam.ac.uk
|
| Phone |
07976413769
|
| Organization name |
MRC Cancer Cell Unit
|
| Department |
MRC/Hutchison Research Centre
|
| Lab |
2.5
|
| Street address |
Box 197, Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0XZ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE18155 |
Malignant Germ Cell Tumors Display Common microRNA Profiles Resulting in Global Changes in Expression of mRNA Targets |
|
