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Sample GSM2706268 Query DataSets for GSM2706268
Status Public on Nov 30, 2017
Title WT10 SHAM 6 WK_mRNA
Sample type RNA
 
Source name Subchondral bone
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 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

Data table header descriptions
ID_REF
VALUE Norm Exp and Cyclic lowess normalized signal intensity

Data table
ID_REF VALUE
A_51_P399985 12.67215954
A_55_P2508138 3.59529285
A_55_P2805880 4.333545831
A_55_P2419483 7.024770879
A_55_P2739683 9.90868758
A_51_P211903 9.281246682
A_66_P121325 5.46300061
A_51_P226429 8.094730089
A_55_P2841743 3.973178739
A_55_P2737159 12.50241882
A_55_P2728466 8.538765069
A_55_P2101526 7.602485673
A_52_P1132414 6.860303493
A_66_P135936 15.40840922
A_55_P2805396 8.331587783
A_55_P2717104 6.978490352
A_55_P2909714 10.88534643
A_55_P2744310 11.97984763
A_52_P83363 5.836802096
A_55_P2091691 12.9456619

Total number of rows: 50815

Table truncated, full table size 1263 Kbytes.




Supplementary file Size Download File type/resource
GSM2706268_Ramaciotti_257480910109_S01_GE1_107_Sep09_1_1.txt.gz 12.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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