Frozen tongue was homogenized in RLT buffer (RNAeasy kit; Qiagen) with a GentleMACS Dissociator (Miltenyi). cDNA was synthesized with a SuperScript III first-strand synthesis system (Invitrogen, Carlsbad, CA). Relative quantification of gene expression was determined by real-time PCR with SYBR green (Quanta BioSciences, Gaithersburg, MD) normalized to Gapdh. Primers were from SA Biosciences (Qiagen). Results were analyzed on a 7300 real-time PCR system (Applied Biosystems, Carlsbad, CA). Knockdown of c-Fos was performed as described in (14 (link)); briefly, 3×105 TR146 cells were serum starved for 24 h and transfected with 37 nM c-Fos siRNA in HiPerFect Reagent (Qiagen) for 2 d. Cells were treated with Clys or IL-17 for 24 h and supernatants analyzed by ELISA.
Protocol detail
c-Fos Knockdown and Cytokine Response
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RNAeasy kit
GentleMACS Dissociator
SuperScript III first-strand synthesis system
SYBR green
7300 real-time PCR system
HiPerFect Reagent
Buffer
Cdna
Cells
Elisa
Frozen
Gapdh
Gene expression
Il 17
Primers
Real time pcr
Serum
Sirna
Sybr green
Synthesis
Tongue
Corresponding Organization : King's College London
Other organizations : Medical College of Wisconsin, Children's Hospital of Wisconsin, University Medical Center of the Johannes Gutenberg University Mainz, Johannes Gutenberg University Mainz, Jena University Hospital, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI)
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Variable analysis
independent variables
- Knockdown of c-Fos
- Treatment with Clys or IL-17
- Relative quantification of gene expression
- Supernatant analysis by ELISA
- Homogenization of frozen tongue in RLT buffer
- CDNA synthesis with SuperScript III first-strand synthesis system
- Real-time PCR with SYBR green normalized to Gapdh
- Serum starvation of TR146 cells for 24 h
- Transfection of TR146 cells with c-Fos siRNA for 2 days
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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