|
| Status |
Public on Dec 01, 2015 |
| Title |
HT29 3D cell culture replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control HT29 cell line cultivated in 2D cell culture
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Control
cell line: HT29
growth condition: 2D
|
| Growth protocol |
For 2D cell culture, cells were cultured in DMEM (HT29 cell line) or RPMI-1640 (DLD1 cell line) cell culture medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2nM glutamine (Gibco), 100 UI/ml penicillin (ROTH) and 0.1 mg/ml streptomycin (ROTH) at 37°C in a humidified atmosphere containing 5% CO2. For 3D cell culture, cells were plated in 0.5 mg/ml laminin rich extracellular matrix (GeltrexTM, Gibco) and cell culture medium upon a layer of agarose ir a 24-well cell culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from harvested cells using GeneJET RNA Purification Kit (ThermoFisher Scientific) according to manufacturer’s instructions. The quantity and quality of RNA were evaluated using Nanodrop 2000c (Thermo Scientific) and Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
1µg of total RNA was used for cDNA synthesis and amplification using MessageTMAmp aRNA kit (Ambion). cRNA labeled with Cy3/Cy5 dyes using Arcturus® TURBO labelingTM CyTM3/CyTM5 Kit (Applied Biosystems).
|
| |
|
| Channel 2 |
| Source name |
HT29 cell line cultivated in 3D cell culture
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Experimental
cell line: HT29
growth condition: 3D
|
| Growth protocol |
For 2D cell culture, cells were cultured in DMEM (HT29 cell line) or RPMI-1640 (DLD1 cell line) cell culture medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2nM glutamine (Gibco), 100 UI/ml penicillin (ROTH) and 0.1 mg/ml streptomycin (ROTH) at 37°C in a humidified atmosphere containing 5% CO2. For 3D cell culture, cells were plated in 0.5 mg/ml laminin rich extracellular matrix (GeltrexTM, Gibco) and cell culture medium upon a layer of agarose ir a 24-well cell culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from harvested cells using GeneJET RNA Purification Kit (ThermoFisher Scientific) according to manufacturer’s instructions. The quantity and quality of RNA were evaluated using Nanodrop 2000c (Thermo Scientific) and Bioanalyzer (Agilent).
|
| Label |
Cy5
|
| Label protocol |
1µg of total RNA was used for cDNA synthesis and amplification using MessageTMAmp aRNA kit (Ambion). cRNA labeled with Cy3/Cy5 dyes using Arcturus® TURBO labelingTM CyTM3/CyTM5 Kit (Applied Biosystems).
|
| |
|
| |
| Hybridization protocol |
825 ng of cRNA labeled with Cy3/Cy5 dyes was prepared for hybridization using Gene Expression Hybridization Kit following manufacturer's protocol (Agilent technologies). Samples were hybridized to Human Gene Expression (v2) 4x44k Oligonucleotide Microarrays (Agilent) for 17 hours at 65°C using HS 400 hybridization station (Tecan).
|
| Scan protocol |
Microarrays were scanned using Tecan LS Reloaded scanner
|
| Description |
Biological replicate 3 of 3. HT29 cells were cultivated under 2D or 3D cell culture conditions for 48 hours, total RNA was extracted and amplified and labeled with Cy3 (2D cell culture) or Cy5 (3D cell culture). For differential gene expression analysis cell grown under 2D conditions served as a control.
|
| Data processing |
Image analysis was performed performed Imagene v9.0 software (BioDiscovery). Raw probe signal values were LOWES normalized
|
| |
|
| Submission date |
Dec 01, 2015 |
| Last update date |
Dec 01, 2015 |
| Contact name |
Vaidotas Stankevicius |
| E-mail(s) |
vaidotas.stankevicius@nvi.lt
|
| Phone |
+37068313680
|
| Organization name |
National Cancer Institute
|
| Lab |
Laboratory of molecullar oncology
|
| Street address |
Santariskiu 1
|
| City |
Vilnius |
| State/province |
Lithuania |
| ZIP/Postal code |
LT-08406 |
| Country |
Lithuania |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE75551 |
Gene expression profile of two colorectal cancer HT29 and DLD-1 cell lines cultivated in 2-dimensional or 3-dimensional cell culture enriched with laminin rich extracellular matrix proteins |
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