|
| Status |
Public on Nov 30, 2017 |
| Title |
SHAM 6 WK_2 |
| Sample type |
RNA |
| |
|
| Source name |
Cartilage
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6
gender: Male
tissue: subchondral bone
time post-surgery (weeks): 6
|
| Treatment protocol |
Post-traumatic OA was surgically induced in 10-12 week old male wild type C57BL6 and Jaffa 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 |
DMM and sham were co-housed with 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-operative medication, were maintained in their pre-operative groups and were allowed unrestricted cage exercise.
|
| 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 cartilage samples were performed on n = 3/group/time point, each consisting of RNA pooled from 3 individual mice. qPCR validation was performed on the same 3 pooled RNA samples and 4 additional biological replicates per time point per group. 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 |
miRNA expression profiling was performed using SurePrint mouse miRNA microarray technology, release 21 (G4859C, Agilent Technologies) at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). Briefly, 70 ng of total RNA was labelled and hybridized using the miRNA microarray System with miRNA complete labelling and Hyb kit version 3.0 (Agilent Technologies) by following manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
miRNA expression profiling was performed using SurePrint mouse miRNA microarray technology, release 21 (G4859C, Agilent Technologies) at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). Briefly, 70 ng of total RNA was labelled and hybridized using the miRNA microarray System with miRNA complete labelling and Hyb kit version 3.0 (Agilent Technologies) by following manufacturer’s instructions.
|
| 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. Limbs randomly allocated to either microarray or histological analysis. Knees taken from the same mouse for both histology and expression profiling. Cartilage and bones samples isolated from the same mouse for parallel expression analyses.
|
| Data processing |
The raw microarray data was background corrected using the Normexp method and normalized with cyclic loess using the bioconductor package Limma (Limma_3.20.9; (Ritchie et al., 2015)). Only probes with 10% greater signal than the negative controls in at least 4 samples were maintained for differential expression analysis. Probes were summarized initially at the probe level then at the gene level using the Limma avereps function, with control probes removed. Differential expression was assessed using moderated t-tests from the Limma package. Results were adjusted for multiple testing using Benjamini-Hochberg method to control for false discovery rate.
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| |
|
| Submission date |
Mar 23, 2017 |
| Last update date |
Nov 30, 2017 |
| 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 |
GPL21265 |
| Series (2) |
| GSE93008 |
Cartilage microRNA dysregulation during the onset and progression of mouse osteoarthritis is independent of aggrecanolysis and overlaps with candidates from end-stage human disease [miRNA] |
| 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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