|
| Status |
Public on Dec 31, 2020 |
| Title |
StyloCSC_2 |
| Sample type |
RNA |
| |
|
| Source name |
Cells sorted from CMT-Stylo through flow cytometry, replicate 2
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: sorted cells from cell line
treatment: non treated
|
| Treatment protocol |
To develop CMT-Star cells from CMT-Stylo, the cells were exposed to increasing concentration of doxorubicin in complete growth media, until the cells became resistant at the level of 100 nM doxorubicin exposure over several months. To isolate cancer stem cells (CSC) from CMT-Stylo and CMT-Star cell respectively, Phycoerythrin (PE) conjugated CD24 antibody (clone M1/69, BD Pharmingen) and Allophycocyanin (APC) conjugated CD44 antibody (clone IM7, BD Pharmingen) were used.
|
| Growth protocol |
Cells were maintained in DMEM with 10% FBS in 5% CO2 at 37 degrees Celcius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from CMT-Stylo cells, CMT-Star cells and their respective cancer stem cells using the RNeasy Plus Mini Kit (Qiagen®) according to manufacturer’s instructions. To ensure DNA-free extracted RNA, a two-step genomic DNA removal was employed which included the manufacturer’s recommendation using the gDNA column spin and additional incubation with RNase-free DNase (Qiagen®) to digest genomic DNA. The concentration and purity of the RNA was determined using nano-spectrophotometer. RNA integrity number (RNA) was determined using a Bioanalyzer, and samples with RIN 7 and above were used for one color microarray hybridization using Agilent platform.
|
| 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 |
0.825 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Hi-RPM Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Canine Gene Expression Microarray (V2:021193) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G4900DA) with 5 μm resolution at wavelengths of 532 nm (Cy3) using the extended dynamic range (10–100 %) setting.
|
| Description |
Cancer stem cells (CSC) isolated from CMT-Stylo cells which are CD44+, CD24-/low and ALDH+.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters with protocol GE1_1200_Jun14 to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 24, 2018 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Gayathri Thevi Selvarajah |
| E-mail(s) |
gayathri@upm.edu.my
|
| Organization name |
University Putra Malaysia
|
| Department |
Faculty of Veterinary Medicine
|
| Street address |
University Putra Malaysia
|
| City |
Serdang |
| State/province |
Selangor |
| ZIP/Postal code |
43400 |
| Country |
Malaysia |
| |
|
| Platform ID |
GPL15379 |
| Series (1) |
| GSE114888 |
Development of gene signatures for doxorubicin resistance and cancer stem cell CD44+, CD24-/low ALD+ isolated from canine mammary adenocarcinoma cell line (mRNA) |
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