|
| Status |
Public on Apr 11, 2013 |
| Title |
Adamts5Dcat cartilage 6wk Post Surgery Biol Rep 3 dye-swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Adamts5Dcat Sham 6wk
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6 Adamts5Dcat
surgery week: 6 week
tissue: cartilage
surgery: Sham
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DMM surgery: Medial menisco-tibial ligament was cut to destabilise the meniscus (DMM) preventing it from performing its normal load-bearing and joint stabilizing function. The subsequent increase in load borne directly by the joint surface initiates breakdown of the articular cartilage. The underlying joint capsule was opened, the anterior fat pad, dissected with forceps to expose the medial menisco-tibial ligament which was cut (confirmed by being able to move the meniscus medially). The joint was flushed with sterile saline and the wound, closed in three layers – joint capsule, subcutaneous tissue (using 8/0 absorbable suture) and skin (using surgical tissue glue). Animals were allowed normal cage exercise immediately after recovery from anaesthesia. The Sham knee joint was also surgically opened as above, the medial menisco-tibial ligament was touched and then the joint closed as described above.
Cartilage collection: Mice were euthanased at 1, 2 or 6 weeks post surgery. Knee joints were dissected and decalcified in RNAlater/EDTA for 72 hours at 4 deg C. Tibial epiphyses were then snap frozen in OCT and stored at -80 deg C. Up to 45 7mM coronal sections were collected within the arthritic lesion zone onto membrane slides. Sections were then laser microdissected to collect the area of cartilage affected by the DMM surgery.
RNA extraction: Total RNA was extracted using Trizol following manufacturer's instructions
RNA amplification: Up to 10ng of total RNA was collected from the microdissected cartilage which and amplified in two rounds using an Ambion Amino Allyl MessageAmp II amplification kit as per the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
RNA labelling: 5µg of aRNA was labelled with Amersham Cy3 or Cy5 dye as per the Ambion Amino Allyl MessageAmp II protocol.
|
| |
|
| Channel 2 |
| Source name |
Adamts5Dcat DMM 6wk
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6 Adamts5Dcat
surgery week: 6 week
tissue: cartilage
surgery: DMM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DMM surgery: Medial menisco-tibial ligament was cut to destabilise the meniscus (DMM) preventing it from performing its normal load-bearing and joint stabilizing function. The subsequent increase in load borne directly by the joint surface initiates breakdown of the articular cartilage. The underlying joint capsule was opened, the anterior fat pad, dissected with forceps to expose the medial menisco-tibial ligament which was cut (confirmed by being able to move the meniscus medially). The joint was flushed with sterile saline and the wound, closed in three layers – joint capsule, subcutaneous tissue (using 8/0 absorbable suture) and skin (using surgical tissue glue). Animals were allowed normal cage exercise immediately after recovery from anaesthesia. The Sham knee joint was also surgically opened as above, the medial menisco-tibial ligament was touched and then the joint closed as described above.
Cartilage collection: Mice were euthanased at 1, 2 or 6 weeks post surgery. Knee joints were dissected and decalcified in RNAlater/EDTA for 72 hours at 4 deg C. Tibial epiphyses were then snap frozen in OCT and stored at -80 deg C. Up to 45 7mM coronal sections were collected within the arthritic lesion zone onto membrane slides. Sections were then laser microdissected to collect the area of cartilage affected by the DMM surgery.
RNA extraction: Total RNA was extracted using Trizol following manufacturer's instructions
RNA amplification: Up to 10ng of total RNA was collected from the microdissected cartilage which and amplified in two rounds using an Ambion Amino Allyl MessageAmp II amplification kit as per the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
RNA labelling: 5µg of aRNA was labelled with Amersham Cy3 or Cy5 dye as per the Ambion Amino Allyl MessageAmp II protocol.
|
| |
|
| |
| Hybridization protocol |
825ng Cy labelled aRNA was prepared and hybridised as per the manufacturer's protocol using Agilent Whole Mouse Genome 44k oligoarray (G4122A).
|
| Scan protocol |
Scanned on an Agilent G2565BA scanner
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1) using GE2_v_5_95_Feb07.
The arrays were analysed using limma in the statistical package R, The two colour arrays were background corrected using norm exp (offest=50) , normalised within arrays with loess, between arrays with Aquantile. Control probes were given a weight of zero for normalisation and biological replicates were accounted for. The linear model was constructed to account for dye, and batch effects (data was generated in 3 batches). Duplicate probes were averaged via Probe name. P values were adjusted for FDR using the Benjamini & Hochberg method
|
| |
|
| Submission date |
Apr 10, 2013 |
| Last update date |
Apr 11, 2013 |
| Contact name |
Lynn Rowley |
| E-mail(s) |
lynn.rowley@mcri.edu.au
|
| Organization name |
Murdoch Childrens Research Institute
|
| Street address |
Flemington Road
|
| City |
Parkville |
| State/province |
Vic |
| ZIP/Postal code |
3435 |
| Country |
Australia |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE45793 |
Differential gene expression in cartilage from the mouse DMM osteoarthritis model |
|
