Isolation of granulocytes: Which transcriptome do we analyze neutrophils or eosinophils?
|Druh||Článek v odborném periodiku|
|Časopis / Zdroj||FOLIA BIOLOGICA|
|Fakulta / Pracoviště MU|
|Klíčová slova||total RNA; gradient centrifugation; cell isolation; magnetic separation; gene expression; expression activity; neutrophils; eosinophils; granulocytes|
|Popis||Isolation of granulocyte cells from blood is necessary for accurate studying of changes in their expression. After gradient 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 (RNAP 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.|