|
| Status |
Public on Sep 09, 2016 |
| Title |
progeroid - control - animal 325 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
liver
|
| Organism |
Mus musculus |
| Characteristics |
genotype: progeroid Ercc1 delta/-
diet: control
tissue: liver
background strain: F1 C57BL6J/FVB
|
| Treatment protocol |
All animals were bred and maintained on AIN93G synthetic pellets (Research Diet Services B.V., Wijk bij Duurstede, The Netherlands). The amount of DR was determined in a prior pilot study but as control food intake of the AL-fed mice was continuously monitored. DR was initiated at 7 weeks of age with 10% (2.1 g/day), when animals almost reached the maximum bodyweight preventing disruption of development of young mice. DR was gradually increased weekly with 10%, until 30% DR (1.6 g/day) was reached at 9 weeks of age. Wild type mice ate on average 3.0 g food per day, resulting in 2.1 g/day for 30% DR. Food was given to the animals just before the start of the dark (active) period to avoid alterations in the biological clock.
|
| Growth protocol |
Ercc1Δ/- mice were obtained by crossing Ercc1Δ/+ (in a pure C57BL6J or FVB background) with Ercc1+/- mice (in a pure FVB or C57BL6J background respectively) to yield Ercc1Δ/- with an F1 C57BL6J/FVB hybrid background. Wild-type F1 littermates were used as controls.All animals used in the studies described in this paper were of the same F1 C57BL6J/FVB hybrid background. Typical unfavorable characteristics, like blindness in an FVB background or deafness in a C57BL6J background, do not occur in this hybrid background. Mice were weighed, visually inspected weekly, and scored for gross morphological and motor abnormalities. Experiments were performed in accordance with the Principles of Laboratory Animal Care and with the guidelines approved by the Dutch Ethical Committee in full accordance with European legislation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver of 11 week old wild type and Ercc1Δ/- mice on AL and DR feeding regimen (n=5). Total RNA was extracted using QIAzol lysis Reagent and miRNAeasy Mini Kits (QIAGEN, Hilden, Germany), following Qiagen protocol. Addition of wash buffers RPE and RWT (QIAGEN) was done mechanically by using the QIAcube (QIAGEN, Hilden, Germany) via the miRNeasy program and stored at -80°C. The concentration of RNA was measured by Nanodrop (Thermo Fisher Scientific) and quality assessment of the RNA using the 2100 Bio-Analyzer (Agilent Technologies, Amstelveen, the Netherlands) following manufacturer’s instructions.
|
| Label |
Hy3
|
| Label protocol |
MicroRNA experiments were conducted at Exiqon Services, Denmark. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 750 ng total RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
liver
|
| Organism |
Mus musculus |
| Characteristics |
sample type: common reference
|
| Treatment protocol |
All animals were bred and maintained on AIN93G synthetic pellets (Research Diet Services B.V., Wijk bij Duurstede, The Netherlands). The amount of DR was determined in a prior pilot study but as control food intake of the AL-fed mice was continuously monitored. DR was initiated at 7 weeks of age with 10% (2.1 g/day), when animals almost reached the maximum bodyweight preventing disruption of development of young mice. DR was gradually increased weekly with 10%, until 30% DR (1.6 g/day) was reached at 9 weeks of age. Wild type mice ate on average 3.0 g food per day, resulting in 2.1 g/day for 30% DR. Food was given to the animals just before the start of the dark (active) period to avoid alterations in the biological clock.
|
| Growth protocol |
Ercc1Δ/- mice were obtained by crossing Ercc1Δ/+ (in a pure C57BL6J or FVB background) with Ercc1+/- mice (in a pure FVB or C57BL6J background respectively) to yield Ercc1Δ/- with an F1 C57BL6J/FVB hybrid background. Wild-type F1 littermates were used as controls.All animals used in the studies described in this paper were of the same F1 C57BL6J/FVB hybrid background. Typical unfavorable characteristics, like blindness in an FVB background or deafness in a C57BL6J background, do not occur in this hybrid background. Mice were weighed, visually inspected weekly, and scored for gross morphological and motor abnormalities. Experiments were performed in accordance with the Principles of Laboratory Animal Care and with the guidelines approved by the Dutch Ethical Committee in full accordance with European legislation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver of 11 week old wild type and Ercc1Δ/- mice on AL and DR feeding regimen (n=5). Total RNA was extracted using QIAzol lysis Reagent and miRNAeasy Mini Kits (QIAGEN, Hilden, Germany), following Qiagen protocol. Addition of wash buffers RPE and RWT (QIAGEN) was done mechanically by using the QIAcube (QIAGEN, Hilden, Germany) via the miRNeasy program and stored at -80°C. The concentration of RNA was measured by Nanodrop (Thermo Fisher Scientific) and quality assessment of the RNA using the 2100 Bio-Analyzer (Agilent Technologies, Amstelveen, the Netherlands) following manufacturer’s instructions.
|
| Label |
Hy5
|
| Label protocol |
MicroRNA experiments were conducted at Exiqon Services, Denmark. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 750 ng total RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA™ microRNA Array 7th Gen [miRBase 19.0] (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat. The hybridization was performed according to the miRCURY LNA™ microRNA Array Instruction manual using a Tecan HS4800™ hybridization station (Tecan, Austria).
|
| Scan protocol |
After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY LNA™ microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ microRNA Array Analysis Software, Exiqon, Denmark).
|
| Description |
EL325
|
| Data processing |
The quantified signals were background corrected (Normexp with offset value 10, see Ritchie et al. 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.
The processed data file (Normalized_signal.txt) contains Log2-ratio for each sample to common reference. Column headers are: Probe ID, MicroRNA annotation, Number of Present calls followed by sample ID found in 'description' field.
|
| |
|
| Submission date |
Jan 26, 2016 |
| Last update date |
Sep 09, 2016 |
| Contact name |
Jeroen Pennings |
| E-mail(s) |
Jeroen.Pennings@rivm.nl
|
| Phone |
+31 88 689 2214
|
| Organization name |
Natl. Inst. Public Health & Environment
|
| Street address |
A. van Leeuwenhoeklaan 9
|
| City |
Bilthoven |
| ZIP/Postal code |
3721MA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL17107 |
| Series (2) |
| GSE77226 |
Lifespan extension by diet restriction in DNA repair deficient progeroid Ercc1Δ/- mice [miRNA] |
| GSE77495 |
Unprecedented life- and healthspan extension and genome preservation by diet restriction in DNA repair deficient progeroid Ercc1 Δ/- mice |
|
