Quantification of Human Swiprosin-1/EFhd2 Expression on Protein and RNA Level.

Abstract

Many Ca2+-binding proteins are differentially regulated under pro-inflammatory conditions in different organs. Specific quantification of RNA and protein expression of those proteins demands validated protocols. Peripheral blood mononuclear cells (PBMC) can mirror an inflammatory status originating from several organs and can therefore be an important diagnostic tool. Swiprosin-1/EFhd2 (EFhd2) is a ~30kDa Ca2+ and F-actin binding, cytoskeletal protein with two central EF hands and a C-terminal coiled-coil domain. Unbiased gene expression analyses and proteomics revealed that EFhd2 is regulated under pro-inflammatory conditions in several cell types and tissues. Here we describe validated protocols to quantify the expression of the human orthologue of Swiprosin-1/EFhd2 on RNA and protein level in PBMC. Both methods reveal that EFhd2 is stronger expressed in monocytes than in B cells of healthy donors. Thus, initial experiments relying on qPCR are likely to provide results with functional relevance. The higher expression of EFhd2 in monocytes could be related to monocyte migration under inflammatory conditions.