|
| Status |
Public on Dec 15, 2020 |
| Title |
OE19 rad 2 |
| Sample type |
RNA |
| |
|
| Source name |
oesophageal adenocarcinoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OE19
cell type: oesophageal adenocarcinoma
treatment: untreated
|
| Treatment protocol |
These are untreated cells. These samples were taken at the same time as a parallel set of samples were treated so that we could investigate the treatment naive RNA levels in these cells that may have contributed to their treatment responses. We collected 2 sets of samples from cells grown to different cell densities appropriate to either radiation treatment or chemotherapuetic drug treatment.
|
| Growth protocol |
All cell lines were cultured using RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) , 50 U/ml penicillin , 50 µg/ml streptomycin , and 100 µg/ml normocin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation from untreated cell samples at time of treatment, including DNase digestion, was performed using the miRNeasy Mini Kit (Qiagen, #217004, Chadstone, Australia) and RNase-free DNase Set (Qiagen, #79254, Chadstone, Australia) as instructed by the manufacturer.
|
| Label |
FAMTM
|
| Label protocol |
For cell line experiments, high throughput QuantStudio™ 12K Flex OpenArray® PCR custom made plates were used for miRNA profiling. These arrays were comprised of a panel of 112 miRNA probes (miRBase version 22 miRNA names, seed sequences, and miRBase accession numbers are in Supplementary file 2) that were selected based upon their abundance in OAC patient samples from our previous study on serum small extracellular vesicle associated miRNAs [94]. For each sample, 3.35 μl of RNA was reverse transcribed using a matching Custom OpenArray® miRNA RT pool (Life Technologies cat # A25630) and the TaqMan® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA Pre-amplifications were carried out with a matching Custom OpenArray® PreAmp pool (Life Technologies cat # 4485255) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593) on 7.5 μl complementary DNA (cDNA)/sample for each pool. The pre-amplified products (4 μl per sample) were diluted at the recommended 1:40 dilution with 156 μl of RNase-free ultra pure water before mixing with TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies cat # 4462164) and loading onto a 384-well TaqMan OpenArray loading plate. PCR runs were performed using a QuantStudio™ 12K Flex Real-Time PCR System.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
ionising radiation
microRNA
|
| Data processing |
Fluorescence data was exported to comma delimited text files and then analysed using R statistical software (version 3.4.3), and Microsoft Excel for Mac (version 16). The cycle threshold (Ct) value for each PCR assay was determined using the qpcR package v1.4 in R (https://cran.r-project.org/web/packages/qpcR/index.html). The relative levels of the miRNAs were determined using the formula 2^(40-Ct), and were normalized using the geometric means of the relative levels of selected HKGs.
Fold-change data set reports differences between cell lines chosen to be treated with cisplatin and 5-FU (treated samples not included here). This is because we chose to use the 2 most sensitive and 2 most resistant cell lines for each treatment (out of the of 8 cell lines that were tested), for our RNA investigations (in this case, miRNAs), and so we used different cell lines for cisplatin and 5-FU.
Normalized data set reports the House Keeping Gene normalised miRNA relative levels
|
| |
|
| Submission date |
Nov 16, 2020 |
| Last update date |
Dec 15, 2020 |
| Contact name |
George Mayne |
| E-mail(s) |
[email protected]
|
| Phone |
+61 08 8204 6088
|
| Organization name |
Flinders University of South Australia
|
| Department |
Surgery
|
| Lab |
3d213
|
| Street address |
Room 3D213, Dept of Surgery, Flinders Medical Center
|
| City |
Bedford Park |
| State/province |
SA |
| ZIP/Postal code |
5042 |
| Country |
Australia |
| |
|
| Platform ID |
GPL22992 |
| Series (2) |
| GSE161149 |
MicroRNA profiling in oesophageal adenocarcinoma cell lines and patient serum samples reveals a role for miR-451a in radiation resistance |
| GSE161585 |
MicroRNA profiling in oesophageal adenocarcinoma cell lines and patient serum samples reveals a role for miR-451a in radiation resistance [baseline RT-PCR] |
|
