|
| Status |
Public on Mar 01, 2021 |
| Title |
CRC 347 RHAMM+ CD44+ |
| Sample type |
RNA |
| |
|
| Source name |
primary human colorectal cancer
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: colorectal cancer
patient id: 347
cell markers: RHAMM+ CD44+
|
| Growth protocol |
5000-10000 numbers of cells from freshly isolated colorectal cancer tissues were directly sorted into RNA extraction dilution by FACS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol (Thermo Fisher Scientific; catalog # 15596026), in accordance with the manufacturer’s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS 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 |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 37°C 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 (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Sep 24, 2019 |
| Last update date |
Mar 02, 2021 |
| Contact name |
Michitaka Nakano |
| Organization name |
Kyushu University
|
| Department |
Department of Medicine and Biosystemic Sciences
|
| Street address |
3-1-1, Maidashi, Higashi-ku
|
| City |
Fukuoka |
| State/province |
Fukuoka |
| ZIP/Postal code |
812-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE137919 |
RHAMM marks proliferative subpopulation within CD44+ human colorectal cancer stem cells |
|
