**4.4. Classification of genes depending on the status of age-related expression**

All spots on the microarray were divided into 16 categories as shown Table 3 after assigning "1" for expressed genes and "0" for unexpressed genes. Here, definitions of "expressed" and "unexpressed" are described in "Materials and methods." Category 1 consists of a total of 6,763 genes expressed in the fetal stage, 12, 20, and 30 weeks of age. Category 2 consists of a total of 7,564 genes expressed at 12, 20, and 30 weeks of age. Category 4 consists of a total

infancy period, but converged with age.

differences were observed except between "fatal stage vs. 20 weeks" and "fatal stage vs. 30 weeks", and between "12 weeks vs. 20 weeks" and "12 weeks vs. 30 weeks" according to an ANOVA analysis using Fisher's Z-transform (P < 0.05). These results suggest that the variation in gene expression intensity within the same age was great in fetal stage and

**Figure 2. Correlation matrix of age-related gene expression.** This color-coded correlation matrix illustrates pairwise correlations between the levels of gene expression in individuals. Probe sets with normalized signals (log-transformed and scaled) were used to calculate correlations between 31 arrays

**4.4. Classification of genes depending on the status of age-related expression** 

All spots on the microarray were divided into 16 categories as shown Table 3 after assigning "1" for expressed genes and "0" for unexpressed genes. Here, definitions of "expressed" and "unexpressed" are described in "Materials and methods." Category 1 consists of a total of 6,763 genes expressed in the fetal stage, 12, 20, and 30 weeks of age. Category 2 consists of a total of 7,564 genes expressed at 12, 20, and 30 weeks of age. Category 4 consists of a total

using Pearson correlation coefficient; signals flagged as "absent" were excluded.

doi:10.1371/journal.pone.0019761.g002

**Figure 3. Age-related correlation coefficients within the same age groups.** Correlation coefficients were calculated between individuals within the same age groups. The bottom and top of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiskers denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. \* p < 0.05, \*\* p < 0.01.

**Figure 4. Age-related correlation coefficients between the different age groups.** Correlation coefficients were calculated between the different age groups. The bottom and top of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiskers denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. \* p < 0.01.

of 3,547 genes expressed after 20 weeks of age. Category 8 consists of a total of 827 genes expressed after 30 weeks of age. Sum of the genes expressed at certain age and those unexpressed (Categories 3, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 15) was 1,051. Its fraction was 5.6% of 18,701 genes (Categories 1, 2, 4, and 8) expressing constantly once they appeared. Category 16 consists of genes unexpressed throughout the breeding period. Figure 5 shows the ratio of the genes belonging to each category.

Whole Blood RNA Analysis, Aging and Disease 109

(GO:0043161, GO:0045059, GO:0019886), while those highly expressed in Category 2 related

**Figure 5. Ratios of categories for groups of the same age.** The ratios of the genes in each category were calculated for groups in the fetal stage and at 12, 20, and 30 weeks of age. Categories are defined in

Expression intensity of immunity gene was examined. Antigen processing and presentation (GO:0019882) and T cell selection (GO0045058) include the major histocompatibility complex (MHC) genes. By presenting antigens, MHC is involved in elimination of bacterial or viral pathogen, rejection of cancer cells, and rejective response on organ transplantation. Also MHC is indispensable in the immune system. Swine leukocyte antigens (SLA) are important immunogens for humoral responses and important mediators of the cellular immune responses through both direct and indirect presentation of peptides to T-cells [12]. SLA includes 6 of classical class I genes (SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, and SLA-8) and 8 of classical class II genes (SLA-DMA, SLA-DMB, SLA-DOA, SLA-DOB1, SLA-DQA, SLA-DQB1, SLA-DRA, and SLA-DRB1) [13-14]. SLA class II lacks DPA1, DPB1, DRB3, DRB4, and DRB 5 in humans. On the Agilent porcine microarray, all of SLA genes except DOA are mounted on 28 spots. Among these, 11 SLA genes fell under Category 1, 1 fell under Category 2, and 1 fell under Category 8. Expression of SLA classical class I and class II genes are shown in Figure 6A and 6B, respectively. Both genes expressed in fatal stage, 12, 20, and

The Agilent porcine microarray had 7 probes with 7 types of interferon and 7 probes for 4 types of interferon receptors. All of 7 interferon genes fell under Category 16. Normally these genes remain unexpressed but expressed upon necessity. In contrast, 1 type of interferon receptor gene fell under Category 1, 3 fell under Category 2, and were expressed

**4.5. Age-related changes in gene expression levels for the immune system** 

to cellular defense and regulation.

doi:10.1371/journal.pone.0019761.g003

30 weeks in an increased manner by age.

Table 3.


Depending on the status of expression, all spots on the microarray can be divided into 16 categories.

Here, "1" represents an expressed gene and "0" represents an unexpressed gene.

**Table 3.** Genes classified into 16 categories according to the status of age-related expression doi:10.1371/journal.pone.0019761.t005

To characterize gene expression in each category, TC Annotator List (Porcine version 14.0 3- 11-10) was downloaded from the TIGR gene Indices. TC Annotator List includes the gene number and the GO terms. Out of 43,603 probes in the Agilent porcine microarray (#G2519F#20109), 6,019 genes bear GO annotation. Microarray cDNA probes were classified by GO terms of "biological processes". Out of all genes, fraction in Categories 1, 2, 4, 8, and 16 were 31%, 20%, 8%, 2%, and 38% respectively.

Then the difference in gene expression between all spots and those in 4 categories (Categories 1, 2, 4, and 8) was examined. GO groups dominantly expressed in Category 1 relates to mitosis (GO:0000070, GO:0000022, GO:0007052, and GO:0007100) and to immune (GO:0043161, GO:0045059, GO:0019886), while those highly expressed in Category 2 related to cellular defense and regulation.

**Figure 5. Ratios of categories for groups of the same age.** The ratios of the genes in each category were calculated for groups in the fetal stage and at 12, 20, and 30 weeks of age. Categories are defined in Table 3.

doi:10.1371/journal.pone.0019761.g003

108 Blood Cell – An Overview of Studies in Hematology

**Category Fetal** 

doi:10.1371/journal.pone.0019761.t005

16 were 31%, 20%, 8%, 2%, and 38% respectively.

the ratio of the genes belonging to each category.

3 1 0 1 1 49

5 1 1 0 1 14 6 0 1 0 1 80 7 1 0 0 1 7

9 1 1 1 0 73 10 0 1 1 0 124 11 1 0 1 0 29

13 1 1 0 0 16

of 3,547 genes expressed after 20 weeks of age. Category 8 consists of a total of 827 genes expressed after 30 weeks of age. Sum of the genes expressed at certain age and those unexpressed (Categories 3, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 15) was 1,051. Its fraction was 5.6% of 18,701 genes (Categories 1, 2, 4, and 8) expressing constantly once they appeared. Category 16 consists of genes unexpressed throughout the breeding period. Figure 5 shows

1 1 1 1 1 6763 genes expressed from fetal

2 0 1 1 1 7564 genes expressed from 12 to 30

4 0 0 1 1 3547 genes expressed from 20 to 30

8 0 0 0 1 827 genes expressed at 30 weeks

12 0 0 1 0 428 genes expressed at 20 weeks

14 0 1 0 0 147 genes expressed at 12 weeks 15 1 0 0 0 84 genes expressed in fetal stage 16 0 0 0 0 23851 genes not expressed from fetal

Depending on the status of expression, all spots on the microarray can be divided into 16 categories.

**of genes Definition** 

stage to 30 weeks

stage to 30 weeks

weeks

weeks

**stage 12 weeks 20 weeks 30 weeks Number** 

**Table 3.** Genes classified into 16 categories according to the status of age-related expression

Here, "1" represents an expressed gene and "0" represents an unexpressed gene.

To characterize gene expression in each category, TC Annotator List (Porcine version 14.0 3- 11-10) was downloaded from the TIGR gene Indices. TC Annotator List includes the gene number and the GO terms. Out of 43,603 probes in the Agilent porcine microarray (#G2519F#20109), 6,019 genes bear GO annotation. Microarray cDNA probes were classified by GO terms of "biological processes". Out of all genes, fraction in Categories 1, 2, 4, 8, and

Then the difference in gene expression between all spots and those in 4 categories (Categories 1, 2, 4, and 8) was examined. GO groups dominantly expressed in Category 1 relates to mitosis (GO:0000070, GO:0000022, GO:0007052, and GO:0007100) and to immune

#### **4.5. Age-related changes in gene expression levels for the immune system**

Expression intensity of immunity gene was examined. Antigen processing and presentation (GO:0019882) and T cell selection (GO0045058) include the major histocompatibility complex (MHC) genes. By presenting antigens, MHC is involved in elimination of bacterial or viral pathogen, rejection of cancer cells, and rejective response on organ transplantation. Also MHC is indispensable in the immune system. Swine leukocyte antigens (SLA) are important immunogens for humoral responses and important mediators of the cellular immune responses through both direct and indirect presentation of peptides to T-cells [12]. SLA includes 6 of classical class I genes (SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, and SLA-8) and 8 of classical class II genes (SLA-DMA, SLA-DMB, SLA-DOA, SLA-DOB1, SLA-DQA, SLA-DQB1, SLA-DRA, and SLA-DRB1) [13-14]. SLA class II lacks DPA1, DPB1, DRB3, DRB4, and DRB 5 in humans. On the Agilent porcine microarray, all of SLA genes except DOA are mounted on 28 spots. Among these, 11 SLA genes fell under Category 1, 1 fell under Category 2, and 1 fell under Category 8. Expression of SLA classical class I and class II genes are shown in Figure 6A and 6B, respectively. Both genes expressed in fatal stage, 12, 20, and 30 weeks in an increased manner by age.

The Agilent porcine microarray had 7 probes with 7 types of interferon and 7 probes for 4 types of interferon receptors. All of 7 interferon genes fell under Category 16. Normally these genes remain unexpressed but expressed upon necessity. In contrast, 1 type of interferon receptor gene fell under Category 1, 3 fell under Category 2, and were expressed until 12 weeks of age. Their signal intensities stayed at constant levels after 12 weeks (Figure 6C).

Whole Blood RNA Analysis, Aging and Disease 111

**5. Gene expression profiles change related to hyperlipidemia** 

blood RNA in miniature pigs.

[23-29].

**RNA** 

Clawn miniature pigs.

To examine the usage of whole blood RNA analysis for the early diagnosis of the disease, we showed transitions in dietary induced hyperlipidemia gene expression profiles of whole

Hyperlipidemia is well recognized as a risk factor for cardiovascular disease (CVD). As diet represents the most important determinant of hyperlipidemia, dietary animal models can be useful for the study of CVD progression [19]. High-fat, high-cholesterol, and high-sugar diets have been shown to induce hyperlipidemia, obesity, and insulin resistance in humans and rodents [20-22]. Dietary-induced hyperlipidemia pig models have also been established

A high-fat and high-cholesterol diet (HFCD) as a typical dietary treatment were used for dietary-induced hyperlipidemia miniature pig models, by using specific pathogen-free (SPF)

Eight 12-week-old, male Clawn miniature pigs were housed individually in cages of 1.5 m2 at the breeder's specific pathogen-free (SPF) facility (Japan Farm Co., Ltd, Kagoshima, Japan) for 27 weeks. Body weights at the beginning of the experiment were 5.1 (2.6) kg (mean (standard deviation; SD)). During this period, 5 pigs were fed with 450 g/day standard dry feed (Kodakara73, Marubeni Nisshin Feed Co., Ltd., Tokyo Japan), and had unlimited access to water (control group). Five pigs were fed a high-fat, high-cholesterol

Almost no changes were observed in fasting plasma triglyceride levels. Fasting plasma total cholesterol concentrations had increased in the HFCD group by week 5 of the feeding period (P<0.001) and were maintained between 350 and 1150 mg/dL from weeks 10–27. Fasting plasma high-density lipoprotein cholesterol (HDL-C) concentrations increased and showed significant differences (P < 0.001) from weeks 10–27. Fasting plasma low-density lipoprotein cholesterol (LDL-C) concentrations also increased and showed significant differences from weeks 5–27. Fasting plasma glucose concentrations remained unchanged.

**5.1. Gene expression profiles of dietary-induced hyperlipidemia for whole blood** 

RNA analyses were conducted on blood samples obtained at weeks 10, 19, and 27 of the feeding periods to characterize the dietary effects on gene expression profiles in whole blood and white blood cells of miniature pigs. Each RNA sample was analyzed by aporcine

Variation in correlation coefficients among individuals on the same diet and between different diet groups was evaluated. Pearson correlation coefficients were used for the correlation analysis. Correlation coefficients for 23 microarrays in total were obtained for a normalized signals log-scale after excluding "absent" spots, definition of "absent" were described in Materials and Methods. A color-coded pairwise correlation matrix is displayed in Figure 7.

gene expression microarray consisting of 43603 oligonucleotide probes.

diet containing 15% lard and 2% cholesterol (HFCD group).

Toll-like receptors (TLRs) are the principal pattern recognition receptors. With this innate immunity, the first immune response is mediated into reserved foreign patterns on recognition. TLRs recognize reserved molecular patterns, start rapid response to protect the host upon infection, and produce signals, such as cytokines and co-stimulatory molecules to activate the adaptive immune system [15-16]. Regulation of the TLR signaling cascade is important for inflammatory responses, innate host defense, and adaptive immune responses [17-18]. Most mammalian species are estimated to have between 10 and 15 types of TLRs. The Agilent porcine microarray has 10 types of TLRs probes. Among these TLRs, 5 of TLR genes fell under Category 2 (expressed until 12 weeks of age), 1 under Category 8, and 4 under Category 16. Their signal intensities remained constant after 12 weeks of age (Figure 6D).

**Figure 6. Signal intensity of major histocompatibility complex (MHC) genes.** (A) Swine leukocyte antigens (SLA) classical class I genes. (B) Swine leukocyte antigens (SLA) classical class II genes. (C) Interferon receptor genes. (D) Toll-like receptor (TLR) genes. Signal intensities were normalized using quantile normalization and log-transformed after excluded signals flagged as "absent." The category numbers are shown in graph legends. Genes in Categories 1, 2, and 4 are shown in the graph. doi:10.1371/journal.pone.0019761.g005
