|
| Status |
Public on May 24, 2022 |
| Title |
HT-29, rep3, PLE-extract |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HT-29 (ATCC® HTB-38™), treated
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: banana passion fruit seeds PLE-extract, 48h
cell line: HT-29
|
| Treatment protocol |
HT-29 cells were seeded at a density of 1.0 ×104 cells/cm2 in T25 cm2 culture flasks and incubated for 24 h at 37 °C with 5% CO2 for cell attachment. Thereafter, cell cultures were treated for 48 h with the seeds PLE-extract at IC50 concentration according to the results of the anti-proliferative activity assay (three replicates). In parallel, cell cultures were treated with extract vehicle (DMSO 0.1% in RPMI 1640) regarded as untreated controls (three replicates).
|
| Growth protocol |
HT-29 colon cancer cells were cultured in a growth medium (RPMI 1640 medium supplemented with Hepes 25 mM, L-glutamine 2.05 mM, fetal bovine serum 10% and gentamicin 50 μg/mL) and were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
300 ng of total RNA were labeled. For each hybridization 600 ng of Cy3 or Cy5 probes were added to 5 ul of 10x Blocking Agent, 1 ul of 25x Fragmentation Buffer and Nuclease free water in a 25 ul reaction, incubated at 60ºC for 30 minutes to fragment RNA and stopped with 25 ul of 2x Hybridization Buffer.
|
| |
|
| Channel 2 |
| Source name |
HT-29 (ATCC® HTB-38™), untreated
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: vehicle, control, 48h
cell line: HT-29
|
| Treatment protocol |
HT-29 cells were seeded at a density of 1.0 ×104 cells/cm2 in T25 cm2 culture flasks and incubated for 24 h at 37 °C with 5% CO2 for cell attachment. Thereafter, cell cultures were treated for 48 h with the seeds PLE-extract at IC50 concentration according to the results of the anti-proliferative activity assay (three replicates). In parallel, cell cultures were treated with extract vehicle (DMSO 0.1% in RPMI 1640) regarded as untreated controls (three replicates).
|
| Growth protocol |
HT-29 colon cancer cells were cultured in a growth medium (RPMI 1640 medium supplemented with Hepes 25 mM, L-glutamine 2.05 mM, fetal bovine serum 10% and gentamicin 50 μg/mL) and were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
300 ng of total RNA were labeled. For each hybridization 600 ng of Cy3 or Cy5 probes were added to 5 ul of 10x Blocking Agent, 1 ul of 25x Fragmentation Buffer and Nuclease free water in a 25 ul reaction, incubated at 60ºC for 30 minutes to fragment RNA and stopped with 25 ul of 2x Hybridization Buffer.
|
| |
|
| |
| Hybridization protocol |
Cy3 and Cy5 samples were mixed, placed on ice, and quickly loaded onto arrays, hybridized at 65ºC for 17 hours in a Hybridization oven rotator and then washed in GE wash buffer 1 at room temperature (1 minute) and in GE Wash Buffer 2 at 37ºC (1 minute). Arrays were dried by centrifugation at 2000 rpm for 2 minutes. Slides were SurePrint G3 Human Gene Expression, 8x60K (G4858A-028004).
|
| Scan protocol |
Slides were scanned on an Agilent G2505C scanner, and images were quantified using Agilent Feature Extraction Software (version 10.7.3.1).
|
| Description |
Biological replicate 3 of 3. Banana passion fruit seeds PLE-extract treated vs untreated control HT-29 cells
|
| Data processing |
Background signal correction using the normexp method with an offset of 50, within array normalization using the LOESS method, and between array scaling using the quantile method were applied using the BioConductor package LIMMA.
|
| |
|
| Submission date |
Jul 02, 2019 |
| Last update date |
May 24, 2022 |
| Contact name |
Alberto Valdés |
| E-mail(s) |
a.valdes@csic.es
|
| Phone |
0034910017991
|
| Organization name |
Instituto de Investigación en Ciencias de la Alimentación
|
| Department |
Bioactividad y Análisis de Alimentos
|
| Lab |
Foodomics
|
| Street address |
C\Nicolás Cabrera, 9
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE133686 |
Foodomics study on the anti-proliferative activity mechanisms on HT-29 colon cancer cells in response to banana passion fruit (Passiflora mollissima) seeds PLE-extract |
|
