|
| Status |
Public on Oct 01, 2011 |
| Title |
US 3 |
| Sample type |
RNA |
| |
|
| Source name |
frozen tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: sarcoma
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. And RNA was prepared using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1ug RNA using the One-Color One-Color Quick Amp Labering kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 25x fragmentation buffer and 10x blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55ul of 2xGE hybridization buffer(HI-RPM) was added to the fragmentation mixture and hybridized to Agilent Whole Human Gene Expression Microarray 4×44K (#014850) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with at room temperature GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10% ).
|
| Description |
Gene expression data from frozen tumor samples
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.1.1.1 (Agilent) using default parameters (GE1-v5_10_Apr08 protocol) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
The algorithm used to normalize data: Threshold raw signals to 1.0. Normalization algorithm 75% Percentile shift. Baseline to median of all samples.
|
| |
|
| Submission date |
Sep 30, 2011 |
| Last update date |
Oct 21, 2011 |
| Contact name |
Tatsuyuki Chiyoda |
| E-mail(s) |
t-chop@sj9.so-net.ne.jp
|
| Organization name |
Keio University School of Medicine
|
| Department |
Obstetrics and Gynecology
|
| Street address |
35 Shinanomachi Shinjuku-ku
|
| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE32507 |
Expression profile of carcinosarcoma (CS), endometrioid adenocarcinoma (EC) and sarcoma (US) of uterine corpus |
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