Abstract
Despite enormous progress and development of high‐throughput methods in genome‐wide mRNA analyses, data on the erythroid transcriptome are still limited, even though they could be useful in medical diagnostics and personalized therapy as well as in research on normal and pathological erythroid maturation. Although obtaining normal and pathological reticulocyte transcriptome profiles should contribute greatly to our understanding of the molecular bases of terminal erythroid differentiation as well as the mechanisms of the hematological diseases, a basic limitation of these studies is the difficulty of efficient reticulocyte RNA isolation from human peripheral blood. The restricted number of possible parallel experiments primarily concern healthy individuals with the lowest number of reticulocytes in the peripheral blood and a low RNA content. In the present study, an efficient method for reticulocyte RNA isolation from healthy individuals and hemolytic anaemia patients is presented. The procedure includes leukofiltration, Ficoll‐Paque gradient centrifugation, Percoll gradient centrifugation, and negative (CD45 and CD61) immunomagnetic separation. This relatively fast and simple four‐stage method was successfully applied to obtain a reticulocyte‐rich population from healthy subjects, which was used to efficiently isolate the high‐quality RNA essential for successful NGS‐based transcriptome analysis.
Highlights
Reticulocytes are non‐nucleated immature red blood cells in peripheral blood of mammalian species
During our studies on hereditary spherocytosis, we identified a family in which two members displayed symptoms of hemolytic anaemia that did not fit the characteristics of the known disease
Each reticulocyte-rich layer was subsequently collected into 50 mL centrifuge tubes and washed twice in PBS-EDTA pH 7.4 buffer (8 min, 550 g at 4°C) to obtain 1 mL of reticulocyte-rich suspension, 50% of which were reticulocytes and ~50% red blood cells
Summary
Reticulocytes are non‐nucleated immature red blood cells in peripheral blood of mammalian species. The detection of reticulocyte RNA with brilliant cresyl blue allows one to distinguish reticulocytes from mature red blood cells as well as enabling the identification of the youngest, highly fluorescent reticulocytes (HFR), supplied early from bone marrow in conditions of increased erythropoietic stimulation, eg, hemolytic anaemia. An example of such conditions is increased hemolysis due to red blood cell pathology. Few procedures for peripheral blood reticulocyte isolation are available in the literature.[7,8] Taken together, developing novel efficient methods for quality reticulocyte RNA isolation constitutes an essential step in further studies on their maturation. The proposed method allows efficient and specific isolation of reticulocyte RNA, which can be successfully used for detection of specific transcripts by both qPCR and RNA‐Seq (RNA sequencing)
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