Thomas, Huw, Moots, Robert ORCID: 0000-0001-7019-6211, Edwards, Steven ORCID: 0000-0002-7074-0552 and Wright, Helen ORCID: 0000-0003-0442-3134
(2015)
Whose gene is it anyway? The effect of preparation purity on neutrophil transcriptome studies.
PLoS One, 10 (9).
e0138982-.
This is the latest version of this item.
Text
PONE-D-15-28620R1_FTC_accepted.docx - Unspecified Access to this file is embargoed until Unspecified. Download (3MB) |
|
Text
journal.pone.0138982.pdf - Published version Download (1MB) |
Abstract
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of “highly pure” neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (
Item Type: | Article |
---|---|
Uncontrolled Keywords: | Neutrophils, Humans, RNA, Messenger, Chemokines, Centrifugation, Density Gradient, Cell Separation, Gene Expression Profiling, Microspheres, Gene Expression Regulation, Adult, Transcriptome, Magnetic Phenomena |
Depositing User: | Symplectic Admin |
Date Deposited: | 28 Sep 2015 09:25 |
Last Modified: | 16 Dec 2022 16:48 |
DOI: | 10.1371/journal.pone.0138982 |
Related URLs: | |
URI: | https://livrepository.liverpool.ac.uk/id/eprint/2028339 |
Available Versions of this Item
-
Whose gene is it anyway? The effect of preparation purity on neutrophil transcriptome studies. (deposited 15 Sep 2015 08:07)
- Whose gene is it anyway? The effect of preparation purity on neutrophil transcriptome studies. (deposited 28 Sep 2015 09:25) [Currently Displayed]