|
| Status |
Public on Sep 11, 2016 |
| Title |
1 wk Saline_3 |
| Sample type |
RNA |
| |
|
| Source name |
Serum
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6
gender: Male
|
| Treatment protocol |
Post-traumatic OA was surgically induced in eighteen 11 week old male wild type C57BL6 mice by unilateral destabilization of the medial meniscus (DMM). Briefly, under isoflourane anaesthesia the medial meniscotibial 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). Joints were flushed with sterile saline to remove any blood prior to separate closure of the joint capsule (simple continuous 8/0 Vicryl), subcutaneous tissue (mattress suture 8/0 Vicryl) and skin (cyanoacrylate). Unilateral sham-operations were also performed in 18 mice, where all procedures were identical except the medial menisco-tibial ligament was visualised but not transected. Antigen induced arthritis (AIA) was induced in eighteen 8 week old male wild type C57BL6 mice. Briefly, mice were immunized on day 0 and day 14 with 200 µg methylated bovine serum albumin (mBSA; Sigma) prepared as an emulsion in combination with Freund’s complete adjuvant (CFA containing 2mg/ml mTB). The mBSA emulsion was administered as two 50l intradermal injections into the dorsal skin in the region of the tail base. Arthritis was induced on day 21 by intra-articular injection of 10µl mBSA (20mg/ml dissolved in sterile saline) into the right knee joint. Control mice (n = 18) were immunised as described above but received an intra-articular injection of 10l saline into the right knee joint.
|
| Growth protocol |
All mice were 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, were maintained in their pre-operative groups and were allowed unrestricted cage exercise. Mice with sham and DMM surgery were co-housed as were saline and AIA animals.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the designated times post arthritis induction mice were deeply anesthetised with 4% isoflurane prior to blood collection. A midline incision was made to enter the abdominal cavity and allow visualization of the heart through the diaphragm. Maximal blood volume (0.5-1ml) was then collected via cardiac puncture (3ml syringe 18 gauge needle), transferred into a sterile tube, and allowed to sit at room temperature for 30 minutes. The tubes were then centrifuged (6000 rpm for 20 min) and the serum harvested and transferred into polypropylene tubes in 0.25ml aliquots and stored at -80°C until all time points were harvested for further processing. Serum was thawed on ice, gently mixed and then microRNA was isolated from 100µl of serum of each mouse using a miRNeasy serum/plasma kit (Qiagen) according to the manufacturers instructions. RNA integrity and miRNA quantity was assessed by capillary electrophoresis with a Bioanalyzer 2100 (Agilent) using a small RNA kit according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
miRNA expression profiling was performed using SurePrint mouse miRNA microarray technology, release 19 (G4872A, Agilent Technologies) at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). Briefly, 10 ng of miRNA 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.
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| |
|
| Hybridization protocol |
miRNA expression profiling was performed using SurePrint mouse miRNA microarray technology, release 19 (G4872A, Agilent Technologies) at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). Briefly, 10 ng of miRNA 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 |
Serum and knee joints from the same mice were collected and analysed for this study
|
| Data processing |
The raw data was processed in statistical language R, using a limma package (limma_3.20.9). Samples were background corrected with Normexp (offset = 50) and normalized with cyclic loess. Only probes that were deemed expressed (10 % greater signal than the negative controls) in at least 5 samples were maintained for differential expression analysis. Array weights were applied to data to down-weight lesser-quality arrays, improving the precision and power of the overall differential expression analysis. Probes were summarised at the miRNA level. Data was adjusted for multiple testing using Benjamini and Hochberg method to control for false discovery rate.
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| |
|
| Submission date |
May 05, 2016 |
| Last update date |
Sep 27, 2016 |
| 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 |
GPL22486 |
| Series (1) |
| GSE81139 |
Utility of circulating serum miRNAs as biomarkers of early cartilage degeneration in animal models of post-traumatic osteoarthritis and inflammatory arthritis |
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