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RNA extraction and quantitative PCR to assay inflammatory gene expression
Output Details
Description
Real-time quantitative PCR (RT-qPCR) is a sensitive assay to determine the production of selected mRNA transcripts in various conditions. We required such an assay to demonstrate the effects of mitochondrial depolarization in the presence of Parkin, since we found that damaged mitochondria recruited the NF-kB effector complex molecules, NEMO and IKKb. We developed this protocol to test levels of NF-kB response genes in a cell model transiently over-expressing Parkin. With this technique we found significant upregulation of key pro-inflammatory genes normalized to a housekeeping gene, Gapdh.
Identifier (DOI)
10.17504/protocols.io.5qpvob15bl4o/v1