Relative quantification was performed to measure MMP-11 expression, using a real-time thermocycler (Eppendorf Mastercycler ep realplex ES). One microliter of cDNA and 0.2 μmol/L of each primers were mixed with SYBR Green RealMasterMix (Eppendorf). S9 and 18S were used as internal reference controls. Primers were designed for mouse MMP-11, S9 and 18S, using the Primer3web version 4.0[19 (link),20 (link)], according to sequences from the GeneBank database. Amplification conditions were: 2 min at 95 °C and three step-cycle of 95 °C for 15 s, 58 °C for 20 s and 68 °C for 20 s, for a total of 40 cycles.
Protocol detail
Quantitative Analysis of MMP-11 Expression
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Cdna
Mmp 11
Mouse
Primers
Sybr green
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Variable analysis
independent variables
- Primers for MMP-11, S9, and 18S
- MMP-11 expression
- S9 and 18S as internal reference controls
- Amplification conditions (2 min at 95 °C, three step-cycle of 95 °C for 15 s, 58 °C for 20 s and 68 °C for 20 s, for a total of 40 cycles)
- S9 and 18S as positive controls
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