Silencing expression with short interfering RNA (siRNA) is a rapid and cost-effective way to analyze the involvement of target genes in a range of biological processes. Here we describe isolation of primary human monocytes from peripheral blood and their in vitro differentiation to macrophages, followed by electroporation with siRNA to silence expression of a disintegrin and metalloproteinase 17 (ADAM17). This enables evaluation of ADAM17's role in cleaving transmembrane proteins, such as its prototypic substrate tumor necrosis factor (TNF), by enzyme-linked immunosorbent assay (ELISA), flow cytometry, or immunoblotting.
Keyphrases
- peripheral blood
- flow cytometry
- poor prognosis
- cancer therapy
- rheumatoid arthritis
- endothelial cells
- high throughput
- hyaluronic acid
- binding protein
- drug delivery
- induced pluripotent stem cells
- gene expression
- pluripotent stem cells
- monoclonal antibody
- single cell
- quantum dots
- sensitive detection
- genome wide identification