|
| Status |
Public on Nov 30, 2017 |
| Title |
WT86 SHAM 1 WK_mRNA |
| Sample type |
RNA |
| |
|
| Source name |
Subchondral bone
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6
gender: Male
tissue: subchondral bone
time post-surgery (weeks): 1
|
| Treatment protocol |
Post-traumatic OA was surgically induced in 10-12 week old male wild type C57BL6 mice by bilateral destabilization of the medial meniscus (DMM), as described previously. Briefly, the medial menisco-tibial ligament was exposed (by medial parapatellar arthrotomy and intrapatellar fat pad elevation, without tissue resection) and transected with curved dissecting forceps by one surgeon (CBL). Bilateral sham-operations were also performed, where the medial menisco-tibial ligament was visualised but not transected. All joints were flushed with sterile saline to remove any blood prior to separate closure of the joint capsule (simple continuous 8/0 polyglactin 910), subcutaneous tissue (mattress suture 8/0 polyglactin 910) and skin (cyanoacrylate).
|
| Growth protocol |
Mice with DMM and sham surgery were co-housed 2-5 animals/30×20×18cm individually-ventilated-cage with filter lids, provided with sterilised bedding and environmental enrichment, maintained at 21-22°C with a 12-hour light/dark cycle, and received water and complete pelleted food ad libitum. Mice received no post-arthritis-induction medication and were allowed unrestricted cage exercise. Animals were sacrificed at 1 and 6 weeks after surgery.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were randomly allocated to groups/harvest time point prior to study commencement using their individual ID numbers. Animals were sacrificed at 1 and 6 weeks after surgery. For each animal, one joint was processed for histology whilst the other was used for microarray expression profiling/qPCR. Microarray experiments on SCB samples were performed on n = 4/group/time point with qPCR validation performed on a different cohort of 4 mice/group/time point. Joint dissection, laser micro-dissection and RNA extraction from mouse cartilage and SCB was performed in a similar fashion to previously described. Briefly, mice were sacrificed 1 and 6 weeks after surgery and the tibial epiphyses were isolated and placed in RNA Later (Life Technologies) containing 10% EDTA for at least 72 hrs at 4°C. After decalcification the samples were washed in DEPC-treated PBS, embedded in OCT and stored at -80°C. Serial 10 µm sagittal cryo-sections were fixed in ethanol and air-dried. Sections of the cartilage and underlying SCB of the medial tibial plateau were laser micro-dissected (Arcturus Bioscience) and collected. Total RNA was extracted from pooled laser micro-dissected sections from each individual mouse joint using TRIzol according to the manufacturer’s instructions
|
| Label |
Cyanine 3-pCp
|
| Label protocol |
mRNA expression profiling was performed using SurePrint mouse mRNA expression V2 microarray technology (G4852B, Agilent Technologies). Briefly, 100ng of total RNA was labelled and hybridized using the low input quick amp WT labelling kit (Agilent Technologies) by following manufacturer’s instructions. Randomized placement of samples on the arrays was performed.
|
| |
|
| Hybridization protocol |
mRNA expression profiling was performed using SurePrint mouse mRNA expression V2 microarray technology (G4852B, Agilent Technologies). Briefly, 100ng of total RNA was labelled and hybridized using the low input quick amp WT labelling kit (Agilent Technologies) by following manufacturer’s instructions. Randomized placement of samples on the arrays was performed.
|
| Scan protocol |
The arrays were scanned on a G2565CA microarray scanner and the features were extracted using Agilent Feature Extraction 12.0.07 software
|
| Description |
Bilateral surgeries performed. Knees taken from the same mouse for both histology and expression profiling.
|
| Data processing |
The raw microarray data was processed in statistical language R, using the limma package (limma_3.20.9) and background corrected with Normexp, normalized within arrays with cyclic loess. This SCB mRNA array data was normalised with mRNA array data from synovial tissue from the same cohort of mice as part of a separate study (GSE99731). Only probes with 10% greater signal than the negative controls in at least 5 samples were maintained for differential expression analysis. Probes were summarised and the data was adjusted for multiple testing using Benjamini-Hochberg method to control for false discovery rate.
|
| |
|
| Submission date |
Jul 18, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
John Bateman |
| Organization name |
Murdoch Childrens Research Institute
|
| Department |
Skeletal Biology
|
| Lab |
John Bateman
|
| Street address |
Flemington Road
|
| City |
Melbourne |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL21163 |
| Series (2) |
| GSE101573 |
Cartilage microRNA dysregulation during the onset and progression of mouse osteoarthritis is independent of aggrecanolysis and overlaps with candidates from end-stage human disease [mRNA] |
| GSE101574 |
Cartilage microRNA dysregulation during the onset and progression of mouse osteoarthritis is independent of aggrecanolysis and overlaps with candidates from end-stage human disease |
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