|
| Status |
Public on Dec 27, 2017 |
| Title |
008 arthroscopic partial meniscectomy |
| Sample type |
RNA |
| |
|
| Source name |
Knee meniscus
|
| Organism |
Homo sapiens |
| Characteristics |
age (years): 50
Sex: Male
bmi (kg/m2): 28.48
|
| Treatment protocol |
A small segment of the injured meniscus was resected from 12 patients during arthroscopic partial meniscectomy and a location-matched segment was obtained from 12 patients during total knee arthroplasty. Tissues were homogenized prior to RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a combination of TRIzol:Chloroform (5:1 ratio) method and Minispin columns (Qiagen). The pulverized tissues were first dissolved in 1 ml of TRIzol reagent (Invitrogen) followed by addition of 0.2 ml of Chloroform. Sample were then vortexed and transferred to phase lock gel tubes. Tubes were centrifuged for 15 min at 4°C. The clear aqueous layer was transferred by decanting of pipetting and 1 volume of 70% RNase-free ethanol was added to precipitate RNA. Finally, RNA was collected using RNeasy spin columns (Qiagen). The quality of the RNA was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
cy5
|
| Label protocol |
20 ng of RNA transcripts were amplified with the use of Sigma-WTA2 kit (Sigma-Aldrich) and then complementary DNA (cDNA) was labelled with Kreatech ULS RNA labeling kit (Kreatech Diagnostics). Briefly, 3 g of cDNA was mixed with Kreatech 10X labeling buffer and Kreatech cyanine 5/DY-ULS. The reaction mixture was incubated at 85oC for 15 min in the dark and then quenched on ice for 3 min. Labelled cDNA was purified with Qiagen purification columns according to the supplier’s protocol (Qiagen).
|
| |
|
| Hybridization protocol |
For hybridization, amplified RNA samples were suspended in Agilent 2X Gene Expression buffer, Agilent 10X blocking agent, and Kreablock. The hybridization solution was applied to human SurePrint G3 Human 8X60K microarrays (Catalog # G4851C, Agilent Technologies) and incubated with streptavidin-cyanine 5 at 65oC for 20h. Washing procedures were performed according to Agilent Gene Expression protocol.
|
| Scan protocol |
Slides were scanned on an Agilent C-class Microarray scanner to detect Cy5 fluorescence as per manufacturer’s specification.
|
| Description |
Gene expression in torn meniscus
|
| Data processing |
Gridding and analysis of images were performed using Feature Extraction software v. 11.5.1.1 (Agilent Technologies). Raw data (probe intensity) were preprocessed by using R package ‘oligo’, and were quantile normalized across all samples. The lowly expressed probeset was further removed by using cutoff at 0.95 quantile probe intensity. R package ‘limma’ was used to build one linear regression model for identifying differentially expressed genes (DEGs) between TKA and APM meniscus, while considering covariance of patients’ age, body mass index and gender at same time.
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| |
|
| Submission date |
May 15, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Bo Zhang |
| E-mail(s) |
[email protected]
|
| Phone |
1-314-362-4757
|
| Organization name |
Washington University School of Medicine
|
| Department |
Developmental Biology
|
| Street address |
660 S. Euclid Avenue
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL20844 |
| Series (1) |
| GSE98918 |
The Impact of Osteoarthritis on the Biology of the Meniscus: |
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