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Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

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 (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.


Expression of chemokine and cytokine transcripts in neutrophils isolated by Polymorphprep (-P) or negative-selection by magnetic beads (-B).(A) Cluster analysis showing expression levels (Log2 RPKM) in neutrophils incubated alone or in the presence of GM-CSF (GM) or TNF. (B-D) Expression levels of chemokine/cytokine genes with the highest level of expression in (B) untreated, (C) TNF and (D) GM-CSF treated neutrophils isolated by Polymorphprep (●) or negative selection (□).
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pone.0138982.g006: Expression of chemokine and cytokine transcripts in neutrophils isolated by Polymorphprep (-P) or negative-selection by magnetic beads (-B).(A) Cluster analysis showing expression levels (Log2 RPKM) in neutrophils incubated alone or in the presence of GM-CSF (GM) or TNF. (B-D) Expression levels of chemokine/cytokine genes with the highest level of expression in (B) untreated, (C) TNF and (D) GM-CSF treated neutrophils isolated by Polymorphprep (●) or negative selection (□).

Mentions: Reports of cytokine and chemokine expression by human neutrophils can be highly dependent upon the purity of neutrophils, which may explain conflicting reports on the expression of, for example, IL-6 and IL-17A by human neutrophils[1,21,22]. We further analysed the transcriptome data of cytokine-treated (GM-CSF, TNFα) neutrophils isolated by Polymorphprep and negative selection, and compared the expression of transcripts for cytokines (interleukins and TNF-family) and chemokines (CCL, CXCL families). Importantly, none of the transcripts were significantly DE in paired Polymorphprep and negative selection samples. We performed cluster analysis of the expression levels of those transcripts that had detectable expression (RPKM>0.3) in at least one dataset (Fig 6A). A breakdown of RPKM values for cytokines with the highest levels of expression across all datasets (lower cluster) can be seen in Fig 6B–6D.


Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

Expression of chemokine and cytokine transcripts in neutrophils isolated by Polymorphprep (-P) or negative-selection by magnetic beads (-B).(A) Cluster analysis showing expression levels (Log2 RPKM) in neutrophils incubated alone or in the presence of GM-CSF (GM) or TNF. (B-D) Expression levels of chemokine/cytokine genes with the highest level of expression in (B) untreated, (C) TNF and (D) GM-CSF treated neutrophils isolated by Polymorphprep (●) or negative selection (□).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581699&req=5

pone.0138982.g006: Expression of chemokine and cytokine transcripts in neutrophils isolated by Polymorphprep (-P) or negative-selection by magnetic beads (-B).(A) Cluster analysis showing expression levels (Log2 RPKM) in neutrophils incubated alone or in the presence of GM-CSF (GM) or TNF. (B-D) Expression levels of chemokine/cytokine genes with the highest level of expression in (B) untreated, (C) TNF and (D) GM-CSF treated neutrophils isolated by Polymorphprep (●) or negative selection (□).
Mentions: Reports of cytokine and chemokine expression by human neutrophils can be highly dependent upon the purity of neutrophils, which may explain conflicting reports on the expression of, for example, IL-6 and IL-17A by human neutrophils[1,21,22]. We further analysed the transcriptome data of cytokine-treated (GM-CSF, TNFα) neutrophils isolated by Polymorphprep and negative selection, and compared the expression of transcripts for cytokines (interleukins and TNF-family) and chemokines (CCL, CXCL families). Importantly, none of the transcripts were significantly DE in paired Polymorphprep and negative selection samples. We performed cluster analysis of the expression levels of those transcripts that had detectable expression (RPKM>0.3) in at least one dataset (Fig 6A). A breakdown of RPKM values for cytokines with the highest levels of expression across all datasets (lower cluster) can be seen in Fig 6B–6D.

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

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 (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.