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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.


Related in: MedlinePlus

Expression levels of non-neutrophil genes in neutrophil preparations.RPKM values for non-neutrophil genes of the antigen targets in the StemCell magnetic bead negative selection isolation kit (A) and (B) non-neutrophil specific genes associated with T and B cells, monocytes, and eosinophils. Neutrophils were either isolated by negative selection (Bead, circle) or by Polymorphprep (Poly, square) from Donor 1 (1) and Donor 2 (2). Neutrophils were treated with 5 ng/mL GM-CSF (shaded grey), 10ng/mL TNFα (shaded white) or untreated (shaded black) for 1h. Horizontal dotted lines represent RPKM expression threshold of 0.3. Horizontal bars represent mean value. (C) The number of read fragments mapping to non-neutrophil genes and (D) neutrophil-specific genes in each library. Data is shown as the average (±SEM) across three treatment conditions (UT, TNFα, GM-CSF) for each Donor and each isolation protocol. (E) The number of mapped reads in each dataset.
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pone.0138982.g004: Expression levels of non-neutrophil genes in neutrophil preparations.RPKM values for non-neutrophil genes of the antigen targets in the StemCell magnetic bead negative selection isolation kit (A) and (B) non-neutrophil specific genes associated with T and B cells, monocytes, and eosinophils. Neutrophils were either isolated by negative selection (Bead, circle) or by Polymorphprep (Poly, square) from Donor 1 (1) and Donor 2 (2). Neutrophils were treated with 5 ng/mL GM-CSF (shaded grey), 10ng/mL TNFα (shaded white) or untreated (shaded black) for 1h. Horizontal dotted lines represent RPKM expression threshold of 0.3. Horizontal bars represent mean value. (C) The number of read fragments mapping to non-neutrophil genes and (D) neutrophil-specific genes in each library. Data is shown as the average (±SEM) across three treatment conditions (UT, TNFα, GM-CSF) for each Donor and each isolation protocol. (E) The number of mapped reads in each dataset.

Mentions: To quantify the efficiency of the negative selection protocol to deplete contaminating cells, transcripts for the cell-specific antigens utilised in the antibody-based negative selection kit were analysed. Of the seven antigens, only transcripts for CD9, CD36, CD2, and CD3 (expressed in eosinophils, monocytes, T-cells/NK cells and T cells, respectively) were detected in any of the samples (Fig 4A). Transcripts for CD36, CD3 and CD2 were detected in all samples from both donors in samples prepared by Polymorphprep, but were absent (or below the RPKM threshold of 0.3) from all samples prepared by negative selection. CD9 transcripts (specific to eosinophils) were highest in the untreated sample from Donor 2 after purification by Polymorphprep (RPKM = 28.9), in line with the observed high numbers of eosinophils present in these preparations. Neutrophils isolated by negative selection had much lower levels of CD9 expression than those isolated by Polymorphprep. However, unlike the other transcripts, levels of CD9 where not entirely absent in samples isolated by negative selection, and were at or around the 0.3 RPKM threshold.


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 levels of non-neutrophil genes in neutrophil preparations.RPKM values for non-neutrophil genes of the antigen targets in the StemCell magnetic bead negative selection isolation kit (A) and (B) non-neutrophil specific genes associated with T and B cells, monocytes, and eosinophils. Neutrophils were either isolated by negative selection (Bead, circle) or by Polymorphprep (Poly, square) from Donor 1 (1) and Donor 2 (2). Neutrophils were treated with 5 ng/mL GM-CSF (shaded grey), 10ng/mL TNFα (shaded white) or untreated (shaded black) for 1h. Horizontal dotted lines represent RPKM expression threshold of 0.3. Horizontal bars represent mean value. (C) The number of read fragments mapping to non-neutrophil genes and (D) neutrophil-specific genes in each library. Data is shown as the average (±SEM) across three treatment conditions (UT, TNFα, GM-CSF) for each Donor and each isolation protocol. (E) The number of mapped reads in each dataset.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138982.g004: Expression levels of non-neutrophil genes in neutrophil preparations.RPKM values for non-neutrophil genes of the antigen targets in the StemCell magnetic bead negative selection isolation kit (A) and (B) non-neutrophil specific genes associated with T and B cells, monocytes, and eosinophils. Neutrophils were either isolated by negative selection (Bead, circle) or by Polymorphprep (Poly, square) from Donor 1 (1) and Donor 2 (2). Neutrophils were treated with 5 ng/mL GM-CSF (shaded grey), 10ng/mL TNFα (shaded white) or untreated (shaded black) for 1h. Horizontal dotted lines represent RPKM expression threshold of 0.3. Horizontal bars represent mean value. (C) The number of read fragments mapping to non-neutrophil genes and (D) neutrophil-specific genes in each library. Data is shown as the average (±SEM) across three treatment conditions (UT, TNFα, GM-CSF) for each Donor and each isolation protocol. (E) The number of mapped reads in each dataset.
Mentions: To quantify the efficiency of the negative selection protocol to deplete contaminating cells, transcripts for the cell-specific antigens utilised in the antibody-based negative selection kit were analysed. Of the seven antigens, only transcripts for CD9, CD36, CD2, and CD3 (expressed in eosinophils, monocytes, T-cells/NK cells and T cells, respectively) were detected in any of the samples (Fig 4A). Transcripts for CD36, CD3 and CD2 were detected in all samples from both donors in samples prepared by Polymorphprep, but were absent (or below the RPKM threshold of 0.3) from all samples prepared by negative selection. CD9 transcripts (specific to eosinophils) were highest in the untreated sample from Donor 2 after purification by Polymorphprep (RPKM = 28.9), in line with the observed high numbers of eosinophils present in these preparations. Neutrophils isolated by negative selection had much lower levels of CD9 expression than those isolated by Polymorphprep. However, unlike the other transcripts, levels of CD9 where not entirely absent in samples isolated by negative selection, and were at or around the 0.3 RPKM threshold.

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.


Related in: MedlinePlus