RNA was isolated with RNeasy kit (QIAGEN, Hilden, Germany) and reverse transcribed to cDNA with Super-Script III reverse transcriptase (QIAGEN). The cDNA was used as template for real time (RT)-PCR using SYBER green PCR Master Mix with primers for ICAM-1, CCL2, TNF-α, IL-1β, IL-6, CXCL1, NOS2, and P2X7.9 (link) Gene expression was assessed using a StepOne Real Time PCR system (ABI 7900 Sequence Detection System). The cDNA samples were run in triplicate. Samples were normalized according to the content of 18S rRNA.
Protocol detail
qPCR Analysis of Inflammatory Markers
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18s rrna
Ccl2
Cdna
Cxcl1
Gene expression
Icam 1
Il 1β
Nos2
Primers
Real time pcr
Reverse transcriptase
Tnf α
Corresponding Organization : Case Western Reserve University
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Variable analysis
independent variables
- RNA isolation method (RNeasy kit)
- Reverse transcription method (Super-Script III reverse transcriptase)
- PCR method (SYBER green PCR Master Mix)
- Primer targets (ICAM-1, CCL2, TNF-α, IL-1β, IL-6, CXCL1, NOS2, and P2X7)
- Gene expression levels of ICAM-1, CCL2, TNF-α, IL-1β, IL-6, CXCL1, NOS2, and P2X7
- Normalization of cDNA samples according to 18S rRNA content
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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