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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.


Correlation between transcriptomes of Polymorphprep and negatively-selected neutrophils.Gene expression (RPKM) in neutrophils isolated by Polymorphprep or negative selection (Beads) for Donors 1 and 2 following incubation with or without the addition of GM-CSF (5ng/mL) or TNFα (10ng/mL) for 1h. Pearson Correlation co-efficient is shown (p<0.01). The solid line indicates a correlation coefficient of r = 1.0 and the dashed lines indicate a fold difference in expression of 2.
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pone.0138982.g003: Correlation between transcriptomes of Polymorphprep and negatively-selected neutrophils.Gene expression (RPKM) in neutrophils isolated by Polymorphprep or negative selection (Beads) for Donors 1 and 2 following incubation with or without the addition of GM-CSF (5ng/mL) or TNFα (10ng/mL) for 1h. Pearson Correlation co-efficient is shown (p<0.01). The solid line indicates a correlation coefficient of r = 1.0 and the dashed lines indicate a fold difference in expression of 2.

Mentions: Having shown that different donors and different neutrophil isolation procedures generate suspensions containing different numbers of contaminating cells, notably eosinophils, it was then necessary to determine how these contaminating cells and isolation techniques contribute to transcriptome studies. Neutrophils were isolated from Donor 1 and Donor 2 blood samples by Polymorphprep and negative selection, and incubated with (or without) inflammatory cytokines (GM-CSF or TNFα) for 1h. RNA was extracted and gene expression quantified using RNA-Seq. Gene expression values (RPKM) across the whole transcriptome correlated significantly between paired neutrophil samples prepared by the two methods (Polymorphprep and negative selection) from each donor, under all three experimental conditions (1h untreated, GM-CSF, TNF) (Fig 3, Pearson Correlation coefficient r is shown for each condition, p<0.01). More outlying transcripts were found in neutrophils from Donor 2, of which several corresponded to eosinophil-specific transcripts, such as CLC, which were expressed at higher levels in the preparation with Polymorphprep.


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)

Correlation between transcriptomes of Polymorphprep and negatively-selected neutrophils.Gene expression (RPKM) in neutrophils isolated by Polymorphprep or negative selection (Beads) for Donors 1 and 2 following incubation with or without the addition of GM-CSF (5ng/mL) or TNFα (10ng/mL) for 1h. Pearson Correlation co-efficient is shown (p<0.01). The solid line indicates a correlation coefficient of r = 1.0 and the dashed lines indicate a fold difference in expression of 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138982.g003: Correlation between transcriptomes of Polymorphprep and negatively-selected neutrophils.Gene expression (RPKM) in neutrophils isolated by Polymorphprep or negative selection (Beads) for Donors 1 and 2 following incubation with or without the addition of GM-CSF (5ng/mL) or TNFα (10ng/mL) for 1h. Pearson Correlation co-efficient is shown (p<0.01). The solid line indicates a correlation coefficient of r = 1.0 and the dashed lines indicate a fold difference in expression of 2.
Mentions: Having shown that different donors and different neutrophil isolation procedures generate suspensions containing different numbers of contaminating cells, notably eosinophils, it was then necessary to determine how these contaminating cells and isolation techniques contribute to transcriptome studies. Neutrophils were isolated from Donor 1 and Donor 2 blood samples by Polymorphprep and negative selection, and incubated with (or without) inflammatory cytokines (GM-CSF or TNFα) for 1h. RNA was extracted and gene expression quantified using RNA-Seq. Gene expression values (RPKM) across the whole transcriptome correlated significantly between paired neutrophil samples prepared by the two methods (Polymorphprep and negative selection) from each donor, under all three experimental conditions (1h untreated, GM-CSF, TNF) (Fig 3, Pearson Correlation coefficient r is shown for each condition, p<0.01). More outlying transcripts were found in neutrophils from Donor 2, of which several corresponded to eosinophil-specific transcripts, such as CLC, which were expressed at higher levels in the preparation with Polymorphprep.

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.