|
| Status |
Public on Dec 10, 2015 |
| Title |
LLC1 3D cell culture replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
LLC1 cell line cultivated in 3D cell culture
|
| Organism |
Mus musculus |
| Characteristics |
sample type: experimental
cell line: LLC1
growth condition: 3D
|
| Growth protocol |
For 2D cell culture, cells were cultured in DMEM 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 enrich in small noncoding RNAs was isolated from harvested cells using mirVana miRNA Isolation Kit (Ambion) 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 |
miRNA labeling was performed using miRNA Complete Labeling and Hyb Kit (Agilent Technologies) according to manufacturer’s instructuctions. In brief, 100 ng of total RNA were defosforilated and directly labeled with Cy3. Samples were dried out and resuspended in Hi-RPM Hybridization Buffer (Agilent Technologies), containing GE Blocking Agent (Agilent, USA), denaturated by heating 5 min in 100°C.
|
| |
|
| Hybridization protocol |
100 ng of RNA labeled with Cy3 dye was prepared for hybridization using Gene Expression Hybridization Kit following manufacturer's protocol (Agilent technologies). Samples were hybridized to Mouse miRNA Expression 8x15k Oligonucleotide Microarrays (Agilent Technologies) for 20 hours at 55°C in rotating hybridization oven.
|
| Scan protocol |
Microarrays were scanned using SureScan Microarray Scanner (Agilent Technologies)
|
| Description |
Biological replicate 2 of 3. LLC1 cells were cultivated under 2D or 3D cell culture conditions for 48 hours, total RNA was extracted and amplified and labeled with Cy3. For differential gene expression analysis cell grown under 2D conditions served as a control.
|
| Data processing |
Image analysis was performed using Feature Extraction v10.7.3.1 (Agilent Technologies). Raw probe value normalization: Experimental Samples were Scaled to mean of Control Samples
|
| |
|
| Submission date |
Dec 09, 2015 |
| Last update date |
Dec 10, 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 |
GPL7732 |
| Series (2) |
| GSE75862 |
miRNA expression profile of mouse Lewis lung carcinoma LLC1 cell line cultivated in 2-dimensional or 3-dimensional cell culture enriched with laminin rich extracellular matrix proteins |
| GSE75864 |
Profile of mouse Lewis lung carcinoma LLC1 cell line cultivated in 2-dimensional or 3-dimensional cell culture enriched with laminin rich extracellular matrix proteins |
|
