In total 24 knees were harvested from 12 mice for gene transcript analysis. Pooling was performed to obtain a suitable amount of cartilage and each experimental unit was a pool of two compartments. The cartilage collected from each individual knee (including a femur and tibia) was treated as one compartment. Total RNA from cartilage in knee joints of mice was isolated with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First strand cDNA was synthesized from total RNA using the PrimeScript RT Reagent kit (Promega Corporation) according to the manufacturer's protocols. mRNA expression of MMP-9 and MMP-13 was measured on a 7500 Real-Rime PCR system with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.). ACTB was used as a reference gene (18 (link)). Gene-specific primer sequences used in the present study are listed in Table I . The expression levels of genes were calculated using the 2-ΔΔCq method (19 (link)).
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Gene Expression Analysis of Murine Knee Cartilage
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Corresponding Organization :
Other organizations : Nanjing Medical University, University of South China, Second Affiliated Hospital of Nanjing Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- MRNA expression of MMP-9
- MRNA expression of MMP-13
- ACTB as a reference gene for normalization
- Positive control: None mentioned
- Negative control: None mentioned
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