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Development and validation of a bovine macrophage specific cDNA microarray.

Jensen K, Talbot R, Paxton E, Waddington D, Glass EJ - BMC Genomics (2006)

Bottom Line: However, the microarray resources available to study these events in livestock animals are limited.The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-gamma and lipopolysaccharide using amplified RNA.A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics & Genomics, Roslin Institute, Roslin, Midlothian, Edinburgh, EH25 9PS, UK. Kirsty.Jensen@bbsrc.ac.uk

ABSTRACT

Background: The response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited.

Results: Here we report the development of a bovine macrophage specific (BoMP) cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate) and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-gamma and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4.

Conclusion: A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

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Related in: MedlinePlus

Scatterplots comparing the differential expression measured by qRT-PCR and microarray analysis for 10 investigated genes at 2 time points after activation. A, average log(differential expression). The numbers indicate the time post activation. B, log(differential expression) of individual animals at 16 hours post activation. The 10 investigated genes were C1R (black circle), CCL2 (red square), CCR1 (green diamond), CD44 (blue triangle), CD86 (orange triangle), CD9 (pink triangle), FN1 (purple triangle), FOS (black cross), IL6 (red cross) and PRNP (green star). The dotted lines denote the lines of equality (y = x).
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Figure 1: Scatterplots comparing the differential expression measured by qRT-PCR and microarray analysis for 10 investigated genes at 2 time points after activation. A, average log(differential expression). The numbers indicate the time post activation. B, log(differential expression) of individual animals at 16 hours post activation. The 10 investigated genes were C1R (black circle), CCL2 (red square), CCR1 (green diamond), CD44 (blue triangle), CD86 (orange triangle), CD9 (pink triangle), FN1 (purple triangle), FOS (black cross), IL6 (red cross) and PRNP (green star). The dotted lines denote the lines of equality (y = x).

Mentions: The correspondence between the qRT-PCR and microarray expression measurements is shown in Figure 1. There is good agreement between the average log(fold changes) for the 10 genes, with an overall correlation of 0.78 (Figure 1A). The means for 6 genes; C1R, CCR1, CD86, FN1, FOS & PRNP, lie close to the line of equality. CD9 and CD44 exhibit 2–3 times higher fold changes by qRT-PCR than by microarray analysis and CCL2 results are 5 and 13 times greater. The fold change was dramatically different for IL6, being 70 and 130 times greater by qRT-PCR analysis than microarray analysis. The correlation between the qRT-PCR and microarray data was further investigated by comparing the results for individual animals. The individual animal values for each gene at 2 hours (data not shown) and 16 hours (Figure 1B) post activation cluster together for 9 of the 10 genes. The CCL2 values have the most spread and one outlying animal influences the difference between the average microarray and qRT-PCR results (Figure 1A). However, the results for IL6 cluster together, illustrating the consistency of the disparity between qRT-PCR and microarray results for this transcript. Investigation of the raw data failed to identify any obvious reason for the lower fold changes measured by microarray analysis, in particular the disparity could not be explained by saturation effects nor excessively low channel intensities (data not shown).


Development and validation of a bovine macrophage specific cDNA microarray.

Jensen K, Talbot R, Paxton E, Waddington D, Glass EJ - BMC Genomics (2006)

Scatterplots comparing the differential expression measured by qRT-PCR and microarray analysis for 10 investigated genes at 2 time points after activation. A, average log(differential expression). The numbers indicate the time post activation. B, log(differential expression) of individual animals at 16 hours post activation. The 10 investigated genes were C1R (black circle), CCL2 (red square), CCR1 (green diamond), CD44 (blue triangle), CD86 (orange triangle), CD9 (pink triangle), FN1 (purple triangle), FOS (black cross), IL6 (red cross) and PRNP (green star). The dotted lines denote the lines of equality (y = x).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590031&req=5

Figure 1: Scatterplots comparing the differential expression measured by qRT-PCR and microarray analysis for 10 investigated genes at 2 time points after activation. A, average log(differential expression). The numbers indicate the time post activation. B, log(differential expression) of individual animals at 16 hours post activation. The 10 investigated genes were C1R (black circle), CCL2 (red square), CCR1 (green diamond), CD44 (blue triangle), CD86 (orange triangle), CD9 (pink triangle), FN1 (purple triangle), FOS (black cross), IL6 (red cross) and PRNP (green star). The dotted lines denote the lines of equality (y = x).
Mentions: The correspondence between the qRT-PCR and microarray expression measurements is shown in Figure 1. There is good agreement between the average log(fold changes) for the 10 genes, with an overall correlation of 0.78 (Figure 1A). The means for 6 genes; C1R, CCR1, CD86, FN1, FOS & PRNP, lie close to the line of equality. CD9 and CD44 exhibit 2–3 times higher fold changes by qRT-PCR than by microarray analysis and CCL2 results are 5 and 13 times greater. The fold change was dramatically different for IL6, being 70 and 130 times greater by qRT-PCR analysis than microarray analysis. The correlation between the qRT-PCR and microarray data was further investigated by comparing the results for individual animals. The individual animal values for each gene at 2 hours (data not shown) and 16 hours (Figure 1B) post activation cluster together for 9 of the 10 genes. The CCL2 values have the most spread and one outlying animal influences the difference between the average microarray and qRT-PCR results (Figure 1A). However, the results for IL6 cluster together, illustrating the consistency of the disparity between qRT-PCR and microarray results for this transcript. Investigation of the raw data failed to identify any obvious reason for the lower fold changes measured by microarray analysis, in particular the disparity could not be explained by saturation effects nor excessively low channel intensities (data not shown).

Bottom Line: However, the microarray resources available to study these events in livestock animals are limited.The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-gamma and lipopolysaccharide using amplified RNA.A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics & Genomics, Roslin Institute, Roslin, Midlothian, Edinburgh, EH25 9PS, UK. Kirsty.Jensen@bbsrc.ac.uk

ABSTRACT

Background: The response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited.

Results: Here we report the development of a bovine macrophage specific (BoMP) cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate) and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-gamma and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4.

Conclusion: A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

Show MeSH
Related in: MedlinePlus