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| Status |
Public on Dec 15, 2020 |
| Title |
Meniscus_26w_SHAM5_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Meniscus_26w_SHAM5_replicate1
|
| Organism |
Sus scrofa |
| Characteristics |
breed: Wuzhishan
tissue: medial meniscus
gender: male
age: 6m
treatment: sham operation
|
| Treatment protocol |
After the surgical sites were shaved and sterilized, the right rear limbs (Ba-Group) were operated upon using a medial parapatellar approach of approximately 10.0cm from the patella to the tuberositas tibiae. The joint was then opened by partly luxating the patella to the lateral. The ACL was then fixed by a clamp and cut at the distal end and resected 1.0cm. Then, the lateral collateral ligament was separated and exposed along the joint line, and a length of 1.0cm was resected. Following washing with 100 ml of sterile saline solution, the articular capsule was then sutured intermittently and independently with 3-0 silk sutures (Aipu Medical Equipment Co., Ltd., Hangzhou, China). The sham operation was carried out on the left knee joint (Aa-Group) with a similar incision to the articular capsule, but no manipulation was made to the ligaments, menisci or articular cartilage.
|
| Growth protocol |
A visit was carried out twice a day after operation. All animals had been treated with penicillin (Kerui Animal Pharmaceutical Co., Ltd, Chengdu, Sichuan Province, China) and tramadol (Duoduo Pharmaceutical Co., Ltd, Jiamusi, Heilongjiang Province, China) for 7 days. After the wound was completely healed, the animal could move freely in the pig pen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from the meniscal tissue was extracted using an E.Z.N.A Total RNA Kit II (Omega Bio-Tek, Inc., USA) according to the manufacturer’s instructions, including a DNase digestion step. The quantity and quality of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE). RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The Labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60°C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA.
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| |
|
| Hybridization protocol |
100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
5Aa
Gene expression of meniscus 26 weeks after sham operation.
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 7 out of 14 samples have flags in Detected (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using the R scripts. GO analysis and Pathway analysis were performed in the standard enrichment computation method.
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| |
|
| Submission date |
Feb 17, 2020 |
| Last update date |
Dec 15, 2020 |
| Contact name |
Fang Ye Han |
| E-mail(s) |
[email protected]
|
| Organization name |
Hainan Provincial People’s Hospital
|
| Department |
Department of Orthopaedic Surgery
|
| Street address |
No. 19 Xiuhua Road
|
| City |
Haikou |
| State/province |
Hainan |
| ZIP/Postal code |
570311 |
| Country |
China |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE145402 |
Experimental study on gene expression profile of medial meniscus after resection of anterior cruciate ligament and lateral collateral ligament |
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