Construction and validation of a Bovine Innate Immune Microarray

Abstract

Background: Microarray transcript profiling has the potential to illuminate the molecular processes that are involved in the responses of cattle to disease challenges. This knowledge may allow the development of strategies that exploit these genes to enhance resistance to disease in an individual or animal population.

Results: The Bovine Innate Immune Microarray developed in this study consists of 1480 characterised genes identified by literature searches, 31 positive and negative control elements and 5376 cDNAs derived from subtracted and normalised libraries. The cDNA libraries were produced from 'challenged' bovine epithelial and leukocyte cells. The microarray was found to have a limit of detection of 1 pg/microg of total RNA and a mean slide-to-slide correlation co-efficient of 0.88. The profiles of differentially expressed genes from Concanavalin A (ConA) stimulated bovine peripheral blood lymphocytes were determined. Three distinct profiles highlighted 19 genes that were rapidly up-regulated within 30 minutes and returned to basal levels by 24 h; 76 genes that were up-regulated between 2-8 hours and sustained high levels of expression until 24 h and 10 genes that were down-regulated. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray analysis. The results indicate that there is a dynamic process involving gene activation and regulatory mechanisms re-establishing homeostasis in the ConA activated lymphocytes. The Bovine Innate Immune Microarray was also used to determine the cross-species hybridisation capabilities of an ovine PBL sample.

Conclusion: The Bovine Innate Immune Microarray has been developed which contains a set of well-characterised genes and anonymous cDNAs from a number of different bovine cell types. The microarray can be used to determine the gene expression profiles underlying innate immune responses in cattle and sheep.

Figure 1

Effect of DNA element quantity on signal from fluorescently labelled random oligonucleotides. The fluorescence intensities of Panomer™ 9 random oligodeoxynucleotides, Alexa Fluor^® 532 conjugate (Molecular Probes, Invitrogen) were measured after hybridisation to β-actin(◆) and GAPDH (■) control elements. These elements were spotted in a gradient of 25, 50, 100, 200 or 400 pg DNA per spot. Error bars represent one standard deviation of the mean.

Figure 2

Effect on the signal reported from DNA elements of varying quantity, length and position within the target transcript. The mean background corrected signal intensity was measured for each β-actin and GAPDH control element on a microarray hybridised with labelled cDNA from ConA stimulated PBLs. (a) Signal reported by β-actin (◆) and GAPDH (■) elements as a function of the quantity of spotted DNA. The error bars denote one standard deviation of the mean. (b) Signal reported by β-actin (◆) and GAPDH (■) elements with DNA lengths ranging from 200 to 1500 bp. (c) Signals reported by β-actin (black) and GAPDH (unshaded) elements of constant DNA length but positioned at the 3'-end, mid-region or 5'-end of the respective target transcripts.

Figure 3

Slide to slide reproducibility of the Bovine Innate Immune Microarray. (a) Schematic diagram of the experimental design. Each arrow represents one microarray slide with the arrow direction indicating the cDNA labelling from Cy5 to Cy3-labelled cDNA. Bovine PBLs were cultured for 24 h with or without ConA (5 μg/ml). (b) An example of slide-to-slide reproducibility depicted as a scatter plot of the signal ratio on slide 1 vs slide 3. The signal ratio was calculated by dividing the background corrected signal for ConA stimulated PBLs by the background corrected signal for unstimulated PBLs.

Figure 4

Estimation of the limit of signal detection for the Bovine Innate Immune Microarray. The Lucidea Universal RNA mix was used in both Cy3 and Cy5 labelling reactions with specific transcripts present at a concentration range of 0.1 to 3000 pg per μg of total sample RNA. The mean background corrected signal, reported from each corresponding Lucidea Calibration control element printed on the microarray is plotted as log₂ (signal) in Cy3 (■) and Cy5 (◆) dye channels. Error bars represent one standard deviation from the mean.

Figure 5

Time course of ConA activation of bovine peripheral blood lymphocytes. (a) Schematic diagram of the experimental design. Each arrow represents one microarray slide with the arrow direction indicating the cDNA labelling from Cy5 to Cy3-label. Bovine PBLs were stimulated with ConA (5 μg/ml) for 0.5, 2, 4, 8 or 24 h. cDNA from the treated cells were compared to cDNA from unstimulated cells. (b) MA plots of microarray data from the ConA activation time course (dye swap replicates are not shown). Labelled cDNA from bovine PBLs treated with ConA (Cy5) were compared to labelled cDNA from unstimulated cells (Cy3). The X-axis shows the total signal intensity for each element present on the microarray (calculated as 1/2*((log₂(Cy5) + log₂(Cy3))). Y-axis shows the log₂ (signal ratio) (log₂(Cy5/Cy3)). Elements with a log₂ (signal ratio) greater than zero represent transcripts which are more abundant in the ConA activated PBL sample.

Figure 6

Clusters of differentially expressed genes from PBLs stimulated with ConA. 252 elements representing 109 unique transcripts were found to be differentially expressed in bovine PBLs during the time course of ConA stimulation. Ten primary K-means clusters were grouped according to similarity into three general profiles; Induction 1, Induction 2, and Suppression 1. These are depicted by the average signal from the elements within the cluster. Y-axis is the background corrected mean signal. The gene symbol for each transcript within the cluster is listed.

Figure 7

Validation of gene expression profiles by quantitative real time RT-PCR (qRT-PCR). The three gene expression profiles discovered by microarray analysis were validated by qRT-PCR with genes representative of each cluster. The log₂ (fold change) was calculated for both microarray data (◆) and qRT-PCR data (■) at each treatment time point relative to that in unstimulated cells. TNF represents the gene expression profile of Induction 1; TNFRSF9, IL2RA and CCL3L1 represent the Induction 2 profile and THBS represents the Suppression 1 profile.

Figure 8

Cross-species hybridisation of an ovine sample to the Bovine Innate Immune Microarray. (a) Schematic diagram of the experiment which used unstimulated ovine and bovine PBLs cultured for 24 h. Each arrow represents one microarray slide where the arrow direction indicates the cDNA labelling from Cy5 to Cy3-label. (b) Scatter plots of microarray data as log₂ (signal). The upper and middle panels show the variation observed when comparing signals from an identical cDNA source which was labelled with both Cy3 and Cy5 dye; bovine/bovine (upper panel) and ovine/ovine (middle panel). The lower panel shows a scatter plot of the log₂ (signal) from ovine PBL cDNA compared to the log₂ (signal) from bovine PBL cDNA.