Limits...
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

Representative spatial heat maps and M-A plots of raw and normalized data. A, spatial heat map for a representative microarray slide before and after normalization. The footnote gives the range of M values for the slide (z-range) and the range excluding the extreme 5% tails of the distribution of M values (saturation) which are excluded from the plot. B, M-A plots for the same representative microarray slide before and after normalization. Green spots denote control genes and the coloured lines represent the curve for each print tip.
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Figure 2: Representative spatial heat maps and M-A plots of raw and normalized data. A, spatial heat map for a representative microarray slide before and after normalization. The footnote gives the range of M values for the slide (z-range) and the range excluding the extreme 5% tails of the distribution of M values (saturation) which are excluded from the plot. B, M-A plots for the same representative microarray slide before and after normalization. Green spots denote control genes and the coloured lines represent the curve for each print tip.

Mentions: Microarray spot intensity and quality data were extracted from the scanned images using the BlueFuse software version II (BlueGnome). Each slide was normalized separately, using the log2-ratios of treatment to reference intensities for all the non-control spots. Normalization and analysis was based on the Limma package of Bioconductor [49], with additional plotting from the Bioconductor Marray package. However, the Limma models were modified at both the normalization and analysis stages. The normalization was a 2-step process of spatial then intensity dependent bias correction. The spatial bias correction was carried out separately for each 2 × 2 group of blocks, by subtracting corresponding row and column means (RC correction, excluding control spots) from each data spot. This simple correction has been used in crop experiments to remove spatial trends and has also been suggested for similar trends across microarrays [50]. The rows and columns may stretch across the whole slide (global) or only across the spots within a block, to more strongly reflect local spatial patterns. The choice of 2 × 2 groups of blocks results in a smoothing of the spatial pattern intermediate between these two. The intensity dependent bias was removed by local block-lowess [51]. The choice of level, global or local, for the two normalization steps was informed by examination of spatial heat diagrams and M-A plots for all possible normalization combinations (Figure 2).


Development and validation of a bovine macrophage specific cDNA microarray.

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

Representative spatial heat maps and M-A plots of raw and normalized data. A, spatial heat map for a representative microarray slide before and after normalization. The footnote gives the range of M values for the slide (z-range) and the range excluding the extreme 5% tails of the distribution of M values (saturation) which are excluded from the plot. B, M-A plots for the same representative microarray slide before and after normalization. Green spots denote control genes and the coloured lines represent the curve for each print tip.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Representative spatial heat maps and M-A plots of raw and normalized data. A, spatial heat map for a representative microarray slide before and after normalization. The footnote gives the range of M values for the slide (z-range) and the range excluding the extreme 5% tails of the distribution of M values (saturation) which are excluded from the plot. B, M-A plots for the same representative microarray slide before and after normalization. Green spots denote control genes and the coloured lines represent the curve for each print tip.
Mentions: Microarray spot intensity and quality data were extracted from the scanned images using the BlueFuse software version II (BlueGnome). Each slide was normalized separately, using the log2-ratios of treatment to reference intensities for all the non-control spots. Normalization and analysis was based on the Limma package of Bioconductor [49], with additional plotting from the Bioconductor Marray package. However, the Limma models were modified at both the normalization and analysis stages. The normalization was a 2-step process of spatial then intensity dependent bias correction. The spatial bias correction was carried out separately for each 2 × 2 group of blocks, by subtracting corresponding row and column means (RC correction, excluding control spots) from each data spot. This simple correction has been used in crop experiments to remove spatial trends and has also been suggested for similar trends across microarrays [50]. The rows and columns may stretch across the whole slide (global) or only across the spots within a block, to more strongly reflect local spatial patterns. The choice of 2 × 2 groups of blocks results in a smoothing of the spatial pattern intermediate between these two. The intensity dependent bias was removed by local block-lowess [51]. The choice of level, global or local, for the two normalization steps was informed by examination of spatial heat diagrams and M-A plots for all possible normalization combinations (Figure 2).

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