 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 19, 2018 |
| Title |
miRNA_DEN, NTL, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
non-tumorous liver (NTL) tissue sample, obtained from diethylnitrosamine (DEN)-treated mouse (DEN experiment)
|
| Organism |
Mus musculus |
| Characteristics |
experiment: DEN
mouse id: DEN63-3
tissue: liver
|
| Treatment protocol |
Mice had free access to chow and water. At the indicated time points, mice were killed and livers were removed. Livers were macroscopically dissected and tumor material, non-tumorous control liver tissue as well as liver tissue from untreated, sex- and age-matched control mice were immediately snap frozen, followed by histopathological confirmation of the tumor tissue.
|
| Growth protocol |
Diethylnitrosamine (DEN) driven liver tumors [PMID: 15329734], Lymphotoxin-α/b-(LTa/b) driven tumors [PMID: 15661974] and Myc-driven liver tumors [PMID: 17589519] were generated as described previously. In brief, for generation of DEN-driven tumors, mice were given intraperitoneal injections of DEN (Sigma) at a dose of 10 mg per kg body weight at 15 d of age [PMID: 26502405]. Mice were observed for development of tumors at 9 months of age. For LTα/β driven tumors, tg1223 mice expressing LTα and -β in a liver-specific manner at high level were followed for 12 months for tumor development [PMID: 19800575]. Finally, for Myc-driven liver tumors, TRE-MYC mice were crossed to LAP-tTA (liver-specific promoter) mice. Animals were maintained on doxycycline (200 mg/kg doxy chow) to suppress MYC expression until 8 weeks of age. Doxycycline was then removed, and mice were followed for evidence of tumor formation [PMID: 19061838].
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA, along with miRNA, was isolated by Directzol RNA Miniprep (Zymo Research, Irvine, CA). RNA integrity was checked on chip analysis (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for analysis only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
|
| Label |
FAMTP
|
| Label protocol |
miRNA expression levels were profiled using the TaqMan OpenArray Rodent MicroRNA Panel (Applied Biosystems; cat.no. 4461105). This platform allows the simultaneous profiling of 750 well-characterized miRNAs (miRBase v15) and 6 controls. Samples were processed according to the manufacturer’s instructions. In brief, for each sample 100ng of total RNA (that included the miRNA fraction) was reverse transcribed using Megaplex RT primers in a set of two pre-defined pools (Pool A and Pool B), each pool contained 381 stem-looped RT primers (375 miRNA targets, 5 positive controls, 1 negative control). The recommended RT thermal cycling conditions were used: i.e. [16°C, 2 min; 42°C, 1 min; 50°C, 1 sec for 40 cycles]; 85°C, 5 min; 4°C hold. The cDNA products (2.5µl) then underwent unbiased PCR preamplification using Megaplex PreAmp Primers in a set of two pools (Pool A and Pool B) of gene-specific forward and reverse primers. Thermal cycling conditions were: 95°C 10 min; 55°C, 2 min; 72°C, 2 min; [95°C, 15 sec, 60°C, 4 min for 12 cycles], 99.9°C, 10 min; 4°C hold. The preamplified cDNA products (4µL) were subsequently 40x diluted in 0.1X TE buffer pH 8.0, and subjected to real-time PCR amplification and analysis using the TaqMan OpenArray Rodent MicroRNA Panel in 3072-well microfluidic (33nL) OpenArray plates that contained dried TaqMan primers and probes for miRNAs and controls.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
07_DEN63-3
|
| Data processing |
OpenArray Digital PCR Software Software (v1.0, Applied Biosystems) was used to analyze and review the amplification plots and threshold cycle (Ct) values obtained. Default analysis settings were used, except that for baseline estimations data from cycles 2-10 were used, and that for some samples the minimum signal value was set at 100 instead of the default value of 300. Data was then exported, and further analyzed in R using the Bioconductor packages HTqPCR [PMID: 19808880] and limma [PMID: 25605792]. In HTqPCR, the minimum and maximum allowable Ct value were set at 8 and 31, respectively.
The qPCR data was normalized by the ΔCt method, using for each sample as internal reference the mean Ct value of 5 endogenous small non-coding RNA controls (U87, Y1, U6, snoRNA135, snoRNA202).
Next, only those miRNAs that were reliably expressed (i.e. Ct<31) in all samples were included for further analysis. This stringent filtering was applied to limit the screening to a set of robustly expressed miRNAs and to increase statistical power, and resulted in the removal of 620 miRNAs. The remaining 130 miRNAs were included in the statistical analysis.
The non-normalized data file reports the Ct values of 846 miRNAs and controls (non-normalized data). Note that some controls are present multiple times. Ct values of 40 indicates N/A.
The file Feature Category reports the Feature Category (i.e. "OK" and "Undetermined") based on expression (Ct-value) of the target. Features having Ct>31 were considered non-expressed, and were thus labeled "Undetermined".
The file normalized reports the ΔCt values of 130 robustly expressed miRNAs, obtained after filtering as described above. (ΔCt normalized data).
|
| |
|
| Submission date |
Aug 09, 2017 |
| Last update date |
Sep 19, 2018 |
| Contact name |
Guido Hooiveld |
| E-mail(s) |
guido.hooiveld@wur.nl
|
| Organization name |
Wageningen University
|
| Department |
Div. Human Nutrition & Health
|
| Lab |
Nutrition, Metabolism & Genomics Group
|
| Street address |
HELIX, Stippeneng 4
|
| City |
Wageningen |
| ZIP/Postal code |
NL-6708WE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL22525 |
| Series (2) |
| GSE102417 |
A comparative miRNA/mRNA analysis in distinct murine liver cancer models reveals miR-193a-5p and NUSAP1 as therapeutic targets in HCC [miRNA] |
| GSE102418 |
A comparative miRNA/mRNA analysis in distinct murine liver cancer models reveals miR-193a-5p and NUSAP1 as therapeutic targets in HCC |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2736280_07_DEN63-3.txt.gz |
34.0 Kb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
|
|
|
|
 |
