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Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs.

Nino-Soto MI, Jozani RJ, Bridle B, Mallard BA - BMC Res Notes (2008)

Bottom Line: Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation.A potential association was found between SLA-DRB1 alleles and inflammation-related genes.Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont, N1G 2W1, Canada. mnino@uoguelph.ca

ABSTRACT

Background: The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells.

Findings: Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation. Genes varied according to the comparison analyzed. Further testing with qPCR revealed the same trend of differential expression for 4 of the genes, although statistical significance was reached for only one.

Conclusion: A new SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.

No MeSH data available.


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Differential transcriptional activity detected by cDNA microarray hybridization and qPCR. Mean fold changes in transcript quantification obtained by cDNA microarray hybridization () and qPCR (□). a) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *04ns01, n = 14) results for the comparison between SLA-DRB1*0502 and *04ns01. b) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *0701, n = 6) results for the comparison between SLA-DRB1*0502 and *0701 alleles. c) Microarray (n = 2 per group) and qPCR (*0701, n = 6; *04ns01, n = 14) results for the comparison between SLA-DRB1*0701 and *04ns01.
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Figure 2: Differential transcriptional activity detected by cDNA microarray hybridization and qPCR. Mean fold changes in transcript quantification obtained by cDNA microarray hybridization () and qPCR (□). a) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *04ns01, n = 14) results for the comparison between SLA-DRB1*0502 and *04ns01. b) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *0701, n = 6) results for the comparison between SLA-DRB1*0502 and *0701 alleles. c) Microarray (n = 2 per group) and qPCR (*0701, n = 6; *04ns01, n = 14) results for the comparison between SLA-DRB1*0701 and *04ns01.

Mentions: Complete and detailed information on microarray experimental protocols, the datasets and the platform were submitted to GEO (accession number GSE7908). Experiments are described according to the MIAME standard [11]. Heterologous hybridizations (bovine probes – porcine targets) were performed to compare the three groups representing defined SLA-DRB1 alleles (n = 2 pigs per group). A loop design was used for reciprocal comparisons. Six comparisons with dye-swap on the same slide were performed for a total of 12 microarrays. Data was analyzed using Acuity 4.0 (Molecular Devices Corp., Sunnyvale, CA, USA) and normalized with the LOWESS algorithm [12]. After normalization, data was filtered based on flags, percentage of saturated pixels, background and intensity uniformity, and signal to noise ratio. The log-ratios of expression were calculated as the base 2 logarithm of the ratios of background-corrected intensity medians of red dye over green dye intensities. A gene was considered to be differentially expressed if it had an absolute value of log-intensity ratio higher or equal to 0.8, representing a fold-change of 1.7 in transcript quantity. Statistical analysis was performed using the Student's t-test with FDR correction for multiple comparisons [13]. Statistical significance was set at p = 0.01. We had previously validated the use of this in-house immune-endocrine bovine microarray with porcine targets [14]. In this study, hybridization resulted in ~90% positive signals (~170 features) in agreement with those previous observations. However, the presence of positive signals of hybridization does not imply that all spots will provide valid results. As previously mentioned, we performed careful filtering to ensure that only consistent data was subject to further analysis. Results from microarray data analysis are summarized in Table 1 and Figure 2. The *0502 allele group showed higher transcriptional activity for CCL4 and IL1B in all comparisons. The *0701 allele group showed less SLA-DQA transcripts in all comparisons. Transcripts amounts for TLR2 and CASP1 were higher in the *0502 and *0701 allele groups respectively, when compared to the *04ns01 group. The small number of genes consistently detected as differentially expressed reflects the tendency of heterologous hybridization to reduce the effective size of a given microarray. Although optimal results are obtained with homologous hybridizations, the use of heterologous microarray hybridization is still a valid approach to assess gene expression profiles given that measures are taken to preserve the quality of the data obtained [15]. In addition, results were verified using qPCR as stated in the next section.


Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs.

Nino-Soto MI, Jozani RJ, Bridle B, Mallard BA - BMC Res Notes (2008)

Differential transcriptional activity detected by cDNA microarray hybridization and qPCR. Mean fold changes in transcript quantification obtained by cDNA microarray hybridization () and qPCR (□). a) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *04ns01, n = 14) results for the comparison between SLA-DRB1*0502 and *04ns01. b) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *0701, n = 6) results for the comparison between SLA-DRB1*0502 and *0701 alleles. c) Microarray (n = 2 per group) and qPCR (*0701, n = 6; *04ns01, n = 14) results for the comparison between SLA-DRB1*0701 and *04ns01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Differential transcriptional activity detected by cDNA microarray hybridization and qPCR. Mean fold changes in transcript quantification obtained by cDNA microarray hybridization () and qPCR (□). a) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *04ns01, n = 14) results for the comparison between SLA-DRB1*0502 and *04ns01. b) Microarray (n = 2 per group) and qPCR (*0502, n = 9; *0701, n = 6) results for the comparison between SLA-DRB1*0502 and *0701 alleles. c) Microarray (n = 2 per group) and qPCR (*0701, n = 6; *04ns01, n = 14) results for the comparison between SLA-DRB1*0701 and *04ns01.
Mentions: Complete and detailed information on microarray experimental protocols, the datasets and the platform were submitted to GEO (accession number GSE7908). Experiments are described according to the MIAME standard [11]. Heterologous hybridizations (bovine probes – porcine targets) were performed to compare the three groups representing defined SLA-DRB1 alleles (n = 2 pigs per group). A loop design was used for reciprocal comparisons. Six comparisons with dye-swap on the same slide were performed for a total of 12 microarrays. Data was analyzed using Acuity 4.0 (Molecular Devices Corp., Sunnyvale, CA, USA) and normalized with the LOWESS algorithm [12]. After normalization, data was filtered based on flags, percentage of saturated pixels, background and intensity uniformity, and signal to noise ratio. The log-ratios of expression were calculated as the base 2 logarithm of the ratios of background-corrected intensity medians of red dye over green dye intensities. A gene was considered to be differentially expressed if it had an absolute value of log-intensity ratio higher or equal to 0.8, representing a fold-change of 1.7 in transcript quantity. Statistical analysis was performed using the Student's t-test with FDR correction for multiple comparisons [13]. Statistical significance was set at p = 0.01. We had previously validated the use of this in-house immune-endocrine bovine microarray with porcine targets [14]. In this study, hybridization resulted in ~90% positive signals (~170 features) in agreement with those previous observations. However, the presence of positive signals of hybridization does not imply that all spots will provide valid results. As previously mentioned, we performed careful filtering to ensure that only consistent data was subject to further analysis. Results from microarray data analysis are summarized in Table 1 and Figure 2. The *0502 allele group showed higher transcriptional activity for CCL4 and IL1B in all comparisons. The *0701 allele group showed less SLA-DQA transcripts in all comparisons. Transcripts amounts for TLR2 and CASP1 were higher in the *0502 and *0701 allele groups respectively, when compared to the *04ns01 group. The small number of genes consistently detected as differentially expressed reflects the tendency of heterologous hybridization to reduce the effective size of a given microarray. Although optimal results are obtained with homologous hybridizations, the use of heterologous microarray hybridization is still a valid approach to assess gene expression profiles given that measures are taken to preserve the quality of the data obtained [15]. In addition, results were verified using qPCR as stated in the next section.

Bottom Line: Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation.A potential association was found between SLA-DRB1 alleles and inflammation-related genes.Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont, N1G 2W1, Canada. mnino@uoguelph.ca

ABSTRACT

Background: The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells.

Findings: Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation. Genes varied according to the comparison analyzed. Further testing with qPCR revealed the same trend of differential expression for 4 of the genes, although statistical significance was reached for only one.

Conclusion: A new SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.

No MeSH data available.


Related in: MedlinePlus