Cargando…
Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients
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 matur...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307756/ https://www.ncbi.nlm.nih.gov/pubmed/30450750 http://dx.doi.org/10.1111/jcmm.13951 |
_version_ | 1783383061121990656 |
---|---|
author | Skulski, Michał Bartoszewski, Rafał Majkowski, Michał Machnicka, Beata Kuliczkowski, Kazimierz Sikorski, Aleksander F. Bogusławska, Dżamila M. |
author_facet | Skulski, Michał Bartoszewski, Rafał Majkowski, Michał Machnicka, Beata Kuliczkowski, Kazimierz Sikorski, Aleksander F. Bogusławska, Dżamila M. |
author_sort | Skulski, Michał |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6307756 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-63077562019-01-04 Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients Skulski, Michał Bartoszewski, Rafał Majkowski, Michał Machnicka, Beata Kuliczkowski, Kazimierz Sikorski, Aleksander F. Bogusławska, Dżamila M. J Cell Mol Med Original Articles 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. John Wiley and Sons Inc. 2018-11-18 2019-01 /pmc/articles/PMC6307756/ /pubmed/30450750 http://dx.doi.org/10.1111/jcmm.13951 Text en © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Articles Skulski, Michał Bartoszewski, Rafał Majkowski, Michał Machnicka, Beata Kuliczkowski, Kazimierz Sikorski, Aleksander F. Bogusławska, Dżamila M. Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title | Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title_full | Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title_fullStr | Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title_full_unstemmed | Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title_short | Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients |
title_sort | efficient method for isolation of reticulocyte rna from healthy individuals and hemolytic anaemia patients |
topic | Original Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307756/ https://www.ncbi.nlm.nih.gov/pubmed/30450750 http://dx.doi.org/10.1111/jcmm.13951 |
work_keys_str_mv | AT skulskimichał efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT bartoszewskirafał efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT majkowskimichał efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT machnickabeata efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT kuliczkowskikazimierz efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT sikorskialeksanderf efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients AT bogusławskadzamilam efficientmethodforisolationofreticulocyternafromhealthyindividualsandhemolyticanaemiapatients |