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(total RNA / grathent centrifugation / cell isolation / magnetic separation / gene expression / expression activity / neutrophils / eosinophils / granulocytes)
Abstract. Isolation of granulocytes from blood is necessary for accurate study of changes in their expression. After grathent centrifugation, we obtain relatively pure granulocyte populations with different ratios of neutrophils and eosinophils. Unfortunately, in many studies in this field the expression results are not set according to the real variability of the granulocyte population. In many cases, the granulocyte population is marked simply as "neutrophils" and the residual population of eosinophils is not considered. Based on our recent study where we tracked the general transcription factor RNA polymerase II, we hypothesized that eosinophils are more transcriptionally active cells than neutrophils. We decided to test our hypothesis on isolated cells because its implications could change our view on many past expression analyses performed on granulocytes. In our experiments, we isolated neutrophils and eosinophils and measured their total RNA production. According to our results, eosinophils produce much more RNA than neutrophils. Therefore, relatively low numbers of highly active eosinophils can markedly affect the whole pool of granulocytic RNA. We want to emphasize that either a detailed description of the cell population or the use of a pure neutrophil population is necessary for the correct interpretation of neutrophil expression analysis results.
Introduction
Various granulocytes are similar in shape, but not so in their different functions. Neutrophils, eosinophils and basophils are distinguished by the presence of cytoplasmic granules. These cells have shrunken and lobulated nuclei with highly condensed chromatin, but they differ in the number of lobes. Neutrophils, the most common type of granulocytes in human blood, contain nuclei composed of three to five lobes. Neutrophils are the first defenders against bacterial, viral, fungal and multicellular pathogens and are the cornerstone of congenital immunity. They also respond to many signals and produce a variety of molecules that play a role in inflammation, tumour immunity, transplant rejection, and autoimmune syndromes. Eosinophils also protect the body against pathogens and parasites. Their granules are packed with huge amounts of RNases that help fight against viral infections (Domachowski and Rosenberg, 2000). Along with basophils, they control the processes associated with allergies and asthma.
To study changes in...