|
| Status |
Public on Jul 14, 2020 |
| Title |
miRNA PDAC fibroblast IMDM Biological remiRNA P 3 |
| Sample type |
RNA |
| |
|
| Source name |
Cultured human fibroblasts from PDAC
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PDAC
media: IMDM
|
| Growth protocol |
Disease-associated fibroblasts from PDAC, CP and PAT were isolated using the outgrowth method, these cells were grown in IMDM supplemented with 10% FBS, 2% L-glutamine, 1% penicillin-streptomycin. In addition, normal PSCs were isolated using the density gradient method and cultured in the same media.These normal PSC were expanded in culture for more than 15 days to achieve adequate yield and purity and referred to as normal-activated (NA) fibroblast. Samples used for array experiments were used from early passages (P1 or P2). Commercial normal fibroblasts (NF) were grown using low serum medium (LSM) (MSC-GRO, Vitro Biopharma) and IMDM supplemented with 10% FBS, 2% L-glutamine, 1% penicillin-streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the high pure miRNA isolation kit (one-column protocol) (Qiagen) for the isolation of total RNA (containing small RNAs) following the manufacturer’s instructions. Purified total RNA was cleaned from genomic DNA using the DNA-freeTM DNA removal kit (ThermoFisher Scientific) following the manufacturer’s instruction.
|
| Label |
Bioitin
|
| Label protocol |
130ng of total RNA (free from DNA) was labelled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix). This process begins with a brief tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample.
|
| |
|
| Hybridization protocol |
Biotin-labelled samples and controls were added to a hybridisation mix and hybridized to Affymetrix GeneChip miRNA 4.0 arrays for 18 hours at 48˚C and 60rpm in an Affymetrix GeneChip hybridisation oven 645.
|
| Scan protocol |
Following hybridisation the arrays were washed and stained on an Affymetrix GeneChip Fluidics Station 450 using a FS450_0002 script and scanned in an Affymetrix Genechip Scanner 3000G.
|
| Description |
Gene expression from disese-associated cultured fibroblasts
|
| Data processing |
Data were RMA normalised in Partek Genomics Suite
|
| |
|
| Submission date |
Dec 05, 2018 |
| Last update date |
Jul 14, 2020 |
| Contact name |
Eithne Costello |
| E-mail(s) |
ecostell@liverpool.ac.uk
|
| Organization name |
University of Liverpool
|
| Department |
Department of Molecular and Clinical Cancer Medicine
|
| Street address |
Ashton Street
|
| City |
Liverpool |
| ZIP/Postal code |
L69 3GE |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19117 |
| Series (2) |
| GSE123377 |
Disease associated fibroblasts from various pancreatic disorders exhibit subtype specific genetic profile [miRNA] |
| GSE123378 |
Disease associated fibroblasts from various pancreatic disorders exhibit subtype specific genetic profile |
|
