Open AccessEfficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients
Author(s) -
Skulski Michał,
Bartoszewski Rafał,
Majkowski Michał,
Machnicka Beata,
Kuliczkowski Kazimierz,
Sikorski Aleksander F.,
Bogusławska Dżamila M.
Publication year - 2019
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.13951
Subject(s) - reticulocyte , transcriptome , rna , biology , immunomagnetic separation , rna extraction , centrifugation , population , percoll , immunology , microbiology and biotechnology , genetics , gene , medicine , gene expression , biochemistry , environmental health
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 ( CD 45 and CD 61) 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.