 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 15, 2024 |
| Title |
Canine adipose-derived mesenchymal stem cells - biological replicate 9 - condition P3 |
| Sample type |
RNA |
| |
|
| Source name |
Isolated and in vitro cultured adipose tissue cells
|
| Organism |
Canis lupus familiaris |
| Characteristics |
Sex: female
cell type: cAD-MSCs
passage: P3
|
| Growth protocol |
The extraction of cAD-MSCs was carried out within a two-hour window from sample collection. This process involved rinsing and cutting the adipose tissue with a scalpel, followed by enzymatic digestion using 0.5% collagenase type 1 (Gibco, Cat. 17100017). The obtained stromal vascular fraction was subsequently placed into T25 cell culture flasks (Nunc, Thermo Fisher Scientific) and cultured in basal media comprising 79% Dulbecco's Modified Eagle Medium with Low Glucose (DMEM Low Glucose) (Gibco, Cat. 31885049), 20% Fetal Bovine Serum (FBS) (Gibco; Cat. 1027010), and 1% Penicillin/Streptomycin (Sigma-Aldrich, Cat. P4333-100ML) at 37°C. Media was replaced after 24 hours, and when adherent cells reached 90% confluence, subculturing was performed using a solution of 0.05% Trypsin and 0.02% EDTA (Sigma-Aldrich, Cat. 59417C-500ML) until proliferation arrest was observed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gene expression analysis using an RT-qPCR array was conducted following total RNA extraction at two-time points during in vitro culture, P3 and P6. The cAD-MSCs reached approximately 90% confluence in T75 cell culture flasks (Nunc, Thermo Fisher Scientific). Subsequently, the cells were detached using a cell scraper (Nunc, Thermo Fisher Scientific) and centrifuged at 235× g for 10 minutes. Total RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Cat. 74106) with 2M dithiothreitol (Sigma-Aldrich, Cat. 43815-1G), following the manufacturer's instructions for approximately 107 cells. The extracted RNA was stored at -80°C until gene expression analysis.
|
| Label |
SYBR Green
|
| Label protocol |
Genomic DNA elimination and complementary DNA synthesis from 800 ng of RNA was accomplished using the RT2 First Strand kit (Qiagen, Cat. 330401) as instructed by the manufacturer. Subsequently, a commercially available RT² Profiler™ PCR Array for Dog Mesenchymal Stem Cells (PAFD-082ZR, Qiagen) with SYBR Green-optimized primer assays (Qiagen, Cat. 330603) was applied, following the manufacturer's instructions for Rotor-Gene Q (Qiagen). The array featured primers targeting 84 genes, categorized as stemness markers, MSC-specific, MSC-associated, and MSC-differentiation genes associated with osteogenesis, adipogenesis, chondrogenesis, myogenesis, and tenogenesis.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Control Group
7-22 3p
|
| Data processing |
Following data acquisition, normalization, and in-depth analysis were conducted using the specialized RT2 Profiler PCR Array Data Analysis software, accessible online at https://dataanalysis2.qiagen.com/pcr (accessed November 10th, 2023). For the normalization it uses the average of five housekeeping genes: B2M, HPRT1 , RPL13A, PGK1, ACTB. The statistical significance was calculated based on a Student’s t-test of the replicate 2^ (- Delta CT) values for each gene in the control and treatment groups. The software automatically appointed a fold change cutoff of 2.0, equal to log2Fold Change ± 1.0.
Fold Change worksheet reports test/control ( P6/P3) ratios.
|
| |
|
| Submission date |
Feb 12, 2024 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Marina Prišlin |
| E-mail(s) |
prislin.marina@gmail.com
|
| Organization name |
Croatian Veterinary Institute
|
| Department |
Virology Department
|
| Street address |
Savska cesta 148
|
| City |
Zagreb |
| ZIP/Postal code |
10000 |
| Country |
Croatia |
| |
|
| Platform ID |
GPL34182 |
| Series (1) |
| GSE255585 |
In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells |
|
| Supplementary data files not provided |
|
|
|
|
 |
