Isolation of granulocytes: which transcriptome do we analyse - neutrophils or eosinophils?

Isolation of granulocytes from blood is necessary for accurate study 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, 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.

Combination of mRNA and protein microarray analysis in complex cell profiling.

UNLABELLED: This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.

Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.

This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.