Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation

Abstract

The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.

Figure 1

Number of genes and expressed sequence tags (ESTs) investigated before (grey bars) and remaining after (white bars) the bioinformatics annotation approach, those remaining being considered acceptable for the microarray. Genes and ESTs assigned to functional categories may be interpreted as a part of more than 1 family.

Figure 2

Variability of gene expression profiles, based on LOWESS normalized median signal intensities. (A) Within-array variation plot, where the X and Y axes represent the median intensity of cyanine-labeled cDNA probes from the same source of RNA but labeled with Cy3 (X) or Cy5 (Y) and hybridized to the same microarray. (B) Between-array variation plot, where the X and Y axes represent the median intensity of Cy3-labeled probes from the same RNA source but hybridized to a different array.

Figure 3A

Validation of microarray data by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) of RNA extracted from unstimulated B cells cultured for 6 h (UN) and B cells stimulated with lipopolysaccharide (LPS) for 6 h, followed by agarose gel electrophoresis, for comparison of the amplified gene for β-actin with those for leukocyte-function-associated antigen 1 (LFA-1), heat-shock protein 70 (HSP70), CD164, caspase 3, Toll-like receptor 4 (TLR4), and invariant chain.

Figure 3B

Ratio of the raw volume fluorescence of the β-actin gene and each target gene in the unstimulated (white bars) and the LPS-stimulated (black bars) B cells, determined from the relative band density on the gel.