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| Status |
Public on Jan 13, 2011 |
| Title |
Murine knee joint_Operated (DMM)_3day_rep3 |
| Sample type |
RNA |
| |
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| Source name |
Whole murine knee joint_destabilised medial meniscus (DMM)_3days
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57B/6; Gender: Male; Age: 10wks; Treatment: operated, destabilisation of mediasl meniscus (DMM) surgery; Tissue: whole knee joint, skin and muscle bulk removed, sample taken using ends of patella and quadriceps tendon as anatomical dissection markers.
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| Biomaterial provider |
Harlen Laboratories; Kennedy Institute of Rheumatology BSU
|
| Treatment protocol |
10 week old male mice were anaesthetized by intra-peritoneal injection of a 1:1:2 mixture of Hypnorm (0.315mg/ml fentanyl citrate and 10mg/ml fluanisone; VetaPharma Ltd, Leeds, UK), Hypnovel (5mg/ml midazolam; Roche), and sterile water for injection, at a dose of 10ml/kg body weight.
The ventral portion of the right (Ipsilateral) knee was shaved and swabbed with iodine to prepare a sterile surgical field. The surgical procedure was carried out with the aid of a dissecting microscope.
A midline incision over the ventral aspect of the knee was made with a no.15 scalpel and the medial compartment entered via a medial para-patellar approach, thus avoiding damage to the medial collateral ligament and minimizing trauma to the quadriceps. The medial menicus was identified and the attachments of its anterior horn to the tibial plateau cut using a 3mm 15deg ophthalmic scalpel. Care was taken to avoid injury to the anterior cruciate ligament and the cartilage surfaces. The anterior third of the meniscus moved freely between the medial femoral condyle and tibial plateau.
The para-pateller window was closed using an absorbable 6/0 Vicryl suture (W9575) and a round-bodied needle to avoid tissue damage. The skin was closed using a 5/0 Ethilon suture (W1601T) with reverse cut needle (Ethilon, USA).
Mice were culled and knees harvested 3 days post surgery.
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| Growth protocol |
Upon receipt from Harlen the mice were kept in an approved animal-care facility, with strict compliance to care and usage protocols. All mice were housed 3-6 per cage in 63x54x30 cm3 standard IV cages and maintained with a 12 h/12h light/dark cycle at an ambient temperature of 21°C. Animals were fed a certified mouse diet (RM3 from Special Dietary Systems, Essex, UK) and water ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ipsilateral (right) legs were removed and ‘debulked’ by removing skin and surrounding muscle. The joint was isolated by sharp severance at the upper and lower edges of the para-pateller tendon ensuring the joint capsule was left intact. These were regarded as reliable, fixed anatomical landmarks.
Harvested knees were snap frozen immediately in liquid nitrogen and kept on dry ice or at -70degC until homogenisation. Frozen tissue was freeze fractured using a pre chilled bio-pulveriser (Stratech Ltd, Suffolk, UK). The resulting fine powder was added to 1ml of TRI® reagent (Trizol) (Helena Biosciences Europe, Sunderland, UK). Trizol extraction protocol was then performed using 1-Bromo-3-Chloropropane (BCP) as a phase separation agent. The upper RNA containing aqueous layer was carefully aspirated and RNA precipitated in an equal volume of 70% ethanol prior to binding on a QIAmp spin column and washed using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, DE) according to the manufacturer’s protocol. RNAase-free DNAase (Qiagen) was used at an intermediate step during the procedure to reduce DNA contamination of the extracted RNA. RNA was eluted in 40ul of Nuclease free water (Promega) and was stored at -80degC until further use.
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| Label |
biotin
|
| Label protocol |
100ng of total RNA from each sample underwent the first cycle of 1st and 2nd strand cDNA synthesis and subsequent in vitro transcription of antisense RNA (cRNA) using the GeneChip WT cDNA Synthesis and Amplification Kit (900673) (Affymetrix, Santa Clara, CA, USA) following manufacturer’s instructions. cRNA was cleaned up using the GeneChip Sample Clean-up Module, Affymetrix (900371). 10ug of cRNA was put into the second cycle of 1st strand cDNA synthesis using random primers and cRNA hydrolised using the GeneChip WT cDNA Synthesis and Amplification Kit, Affymetrix (900673). cDNA was cleaned up using the GeneChip Sample Clean-up Module, Affymetrix (900371) and 5.5ug single stranded cDNA fragmented and labeled using the GeneChip WT Terminal Labeling Kit, Affymetrix (900671).
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| Hybridization protocol |
The resulting biotin-labeled cDNA was hybridised to the GeneChip Mouse Gene 1.0 ST Array (MoGene_1-0_st-v1) (Affymetrix). Hybridisation was carried out using a GeneChip 450 fluidics station and GeneChip Hybridization Oven 640 at UCL Genomics Department (University College London, London, UK) using a standard protocol.
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| Scan protocol |
Genechips were scanned using the GeneChip® Scanner 3000 7G (Affymetrix) at UCL Genomics Department (University College London, London, UK).
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| Data processing |
Data were processed using aligent Genespring GX software and Robust multiarray average (RMA-16) normalisation algorithim. Default settings for Affymetrix single probe genechips were used. Each sample was assigned the parameter 'time' (time post surgery) and 'treatment' (type of surgery). Signal intensity values for all experimental replicates were average and used for analysis.
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| Submission date |
Jan 05, 2011 |
| Last update date |
Jan 13, 2011 |
| Contact name |
Annika Burleigh |
| E-mail(s) |
[email protected]
|
| Phone |
02083834432
|
| Organization name |
Imperial College London
|
| Department |
Kennedy Institute of Rheumatology
|
| Lab |
Cell Signaling
|
| Street address |
65 Aspenlea Road
|
| City |
London |
| ZIP/Postal code |
W6 8LH |
| Country |
United Kingdom |
| |
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| Platform ID |
GPL6246 |
| Series (1) |
| GSE26475 |
Microarray expression data from whole murine knee joints at early time points post surgery in the destabilization of medial meniscus (DMM) model of OA |
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