Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

Additional File 1:

Statistical analysis and statistical pattern matching results (Statistical_Analysis_and_Statistical_Pattern_Matching_Results.txt). Results from statistical pattern matching are reported as a .txt file. Results are reported for all 14,129 genes considered for statistical analysis, but genes that were not differentially expressed in any tissue are assigned a pattern of zero. Transcript_ID is an identifier associated with the gene analyzed. The gene assignment entry was taken from the Affymetrix annotation file and displays which gene is associated with the given transcript ID. Tissue p, physiological state p, and tissue*physiological state p correspond to the p-values associated with the tissue main effect, physiological state main effect, and the interaction, respectively. p Liver, p duodenum, p jejunum, and p ileum represent the p-values associated with the corresponding simple effects Pattern indicates in which pattern a gene is detected. A zero indicates no differential expression. Otherwise, patterns are assigned as described in the Methods. Ratio liver, ratio duodenum, ratio jejunum, and ratio ileum represent the ratio of the mean of samples from lactating animals to the mean of samples from controls utilizing the untransformed microarray data.

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Additional File 2:

Benjamini-Hochberg false discovery rates (Benjamini_Hochberg_False_Discovery_Rates.txt). False Discovery Rate corrections [81] for all genes studied is presented in .txt format. Transcript_ID is an Affymetrix Identifier for each gene. False discovery rates are provided for the tissue*physiological state interaction term and the overall physiological state effect. Q-values were calculated as the number of false positives expected by chance (uncorrected p* total number of tests) divided by the total number of results with an equal or lower p-value.

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Additional File 3:

Histograms of p-values (Histograms_of_p_values.ppt). Histograms for A) tissue effect p-values, B) physiological state effect p-values, C) physiological state*tissue interaction p-values, and pairwise comparison p-values for D) the liver, E) duodenum, F) jejunum, and G) ileum presented as a .ppt file. While a large tissue effect was observed, a visible treatment effect (control vs. lactation) was also observed.

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Additional File 4:

Volcano plots (Volcano_plots.doc). Volcano plots comparing the log₂ fold changes (reported as mean untransformed lactating intensity divided by untransformed mean control intensity) against the calculated pairwise comparison p-value for each individual tissue in .doc format. Volcano plots are for A) Liver, B) Duodenum, C) Jejunum, and D) Ileum. Each tissue responded differently to lactation. The blue line indicates the significance cutoff of p < 0.01. The number of differentially expressed genes were 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum, when an overall treatment main effect p-value cutoff of p < 0.05 was incorporated. Of particular note is a series of genes that were strongly downregulated in the duodenum (Additional File 1 pattern -100; discussed in Results.)

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Additional File 5:

DAVID output (DAVID_output_file.txt). DAVID was utilized to test specific patterns from statistical pattern matching. Patterns were chosen based on similarity between tissues and were "Up in All Tissues", "Down in all tissues", "Up in all parts of small intestine", "Down in all parts of small intestine", "Up only in liver", "Down only in liver", and "Down only in duodenum". Table truncated from output in DAVID. Genes listed by Affymetrix transcript cluster ID, which may be referenced in Additional File 1.

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Additional File 6:

Genes with decreased mRNA in all tissues (Genes_with_decreased_mrna_all_tissues.doc). To be considered part of a grouping, genes must have a physiologic state p < 0.05 and at least one tissue simple effect p < 0.01. Reported p-values are tissue simple effect p-values and represent the comparison between Lactation and Control in the corresponding tissue. For the purposes of assigning patterns, the significance cutoff for the remaining tissue simple effect was set to p < 0.05. Abbreviations used as in Table 1. *Gene is at the Extended confidence level.

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Additional File 7:

Biosynthesis of sterols in liver (Biosynthesis_of_sterols_in_liver.jpg). Image from IPA representing the "Biosynthesis of Sterols" in the liver as a .jpg file. Numbering system for enzymes in the pathway is taken from KEGG [77]. Components of the cholesterol biosynthetic pathway include 1.1.1.34 (Hmgcr), 2.7.1.36 (Mvk), 2.7.4.2 (Pmvk), 4.1.1.83 (Mvd), 5.3.3.2 (Idi1), 2.5.1.21 (Fdft1), and 1.14.99.7 (Sqle), 5.4.99.7 (Lss), and 1.3.1.21 (Dhcr7). Red shading indicates increased mRNA during lactation from the corresponding gene.

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Additional File 8:

Biosynthesis of sterols in duodenum (Biosynthesis_of_sterols_in_duodenum.jpg). Image from IPA representing the "Biosynthesis of Sterols" in the duodenum as a .jpg file. Numbering system for enzymes in the pathway is taken from KEGG [77]. Components of the cholesterol biosynthetic pathway include 1.1.1.34 (Hmgcr), 2.7.1.36 (Mvk), 2.7.4.2 (Pmvk), 4.1.1.83 (Mvd), 5.3.3.2 (Idi1), 2.5.1.21 (Fdft1), and 1.14.99.7 (Sqle), 5.4.99.7 (Lss), and 1.3.1.21 (Dhcr7). Red shading indicates increased mRNA during lactation from the corresponding gene.

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Additional File 9:

Biosynthesis of sterols in jejunum (Biosynthesis_of_sterols_in_jejunum.jpg). Image from IPA representing the "Biosynthesis of Sterols" in the jejunum as a .jpg file. Numbering system for enzymes in the pathway is taken from KEGG [77]. Components of the cholesterol biosynthetic pathway include 1.1.1.34 (Hmgcr), 2.7.1.36 (Mvk), 2.7.4.2 (Pmvk), 4.1.1.83 (Mvd), 5.3.3.2 (Idi1), 2.5.1.21 (Fdft1), and 1.14.99.7 (Sqle), 5.4.99.7 (Lss), and 1.3.1.21 (Dhcr7). Red shading indicates increased mRNA during lactation from the corresponding gene.

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Additional File 10:

Biosynthesis of sterols in ileum (Biosynthesis_of_sterols_in_ileum.jpg). Image from IPA representing the "Biosynthesis of Sterols" in the ileum as a .jpg file. Numbering system for enzymes in the pathway is taken from KEGG [77]. Components of the cholesterol biosynthetic pathway include 1.1.1.34 (Hmgcr), 2.7.1.36 (Mvk), 2.7.4.2 (Pmvk), 4.1.1.83 (Mvd), 5.3.3.2 (Idi1), 2.5.1.21 (Fdft1), and 1.14.99.7 (Sqle), 5.4.99.7 (Lss), and 1.3.1.21 (Dhcr7). Red shading indicates increased mRNA during lactation from the corresponding gene.

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Additonal File 11:

Genes regulated by Srebp proteins (Genes_Regulated_by_Srebp_proteins.doc). Genes that increase expression in Srebp-1a overexpressing mice and Srebp-2 overexpressing mice, and decrease expression in Scap knockout mice [34]. Overrepresentation analysis showed that genes in this list occurred more frequently than expected by chance in the lists of differentially expressed genes (p < 1 × 10^-4 in each tissue.) Abbreviations used as in Table 1. *Gene is at the Extended confidence level.

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Additional File 12:

Members of the Slc superfamily (Slcs.doc). Table displaying members of the Slc superfamily. The p < 0.01 column indicates in which tissues a change was detected. Abbreviations are as defined for Table 1. * Gene is at the Extended level of confidence. ^aSubstrates taken from the SLC tables database (http://www.bioparadigms.org/ webcite)

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Additional File 13:

Canonical pathways in the liver (Liver_canonical_pathways.txt). This file contains the canonical pathways in IPA and the corresponding -log₁₀ p-values and the individual molecules that were detected as being in the list of overrepresented genes and part of each pathway (listed in the "molecules" column.) FDRs for the individual pathways are shown in a separate series of rows underneath the -log₁₀ p-values. This table shows results utilizing the list of differentially expressed genes in the liver.

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Additional File 14:

Canonical pathways in the duodenum (Duodenum_canonical_pathways.txt). This file contains the canonical pathways in IPA and the corresponding -log₁₀ p-values and the individual molecules that were detected as being in the list of overrepresented genes and part of each pathway (listed in the "molecules" column.) FDRs for the individual pathways are shown in a separate series of rows underneath the -log₁₀ p-values. This table shows results utilizing the list of differentially expressed genes in the duodenum.

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Additional File 15:

Canonical pathways in the jejunum (Jejunum_canonical_pathways.txt). This file contains the canonical pathways in IPA and the corresponding -log₁₀ p-values and the individual molecules that were detected as being in the list of overrepresented genes and part of each pathway (listed in the "molecules" column.) FDRs for the individual pathways are shown in a separate series of rows underneath the -log₁₀ p-values. This table shows results utilizing the list of differentially expressed genes in the jejunum.

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Additional File 16:

Canonical pathways in the ileum (Ileum_canonical_pathways.txt). This file contains the canonical pathways in IPA and the corresponding -log₁₀ p-values and the individual molecules that were detected as being in the list of overrepresented genes and part of each pathway (listed in the "molecules" column.) FDRs for the individual pathways are shown in a separate series of rows underneath the -log₁₀ p-values. This table shows results utilizing the list of differentially expressed genes in the ileum.

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Additional File 17:

Thyroid pathway in liver (Liver_thyroid_pathway.jpg). Images from IPA for the TR/RXR pathway for the liver. Red shading indicates increased mRNA amounts of the respective gene during lactation, and green shading indicates decreased amounts of mRNA.

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Additional File 18:

Thyroid pathway in duodenum (Duodenum_thyroid_pathway.jpg). Images from IPA for the TR/RXR pathway for the duodenum. Red shading indicates increased mRNA amounts of the respective gene during lactation, and green shading indicates decreased amounts of mRNA.

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Additional File 19:

Thyroid pathway in jejunum (Jejunum_thyroid_pathway.jpg). Images from IPA for the TR/RXR pathway for the jejunum. Red shading indicates increased mRNA amounts of the respective gene during lactation, and green shading indicates decreased amounts of mRNA.

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Additional File 20:

Thyroid pathway in ileum (Ileum_thyroid_pathway.jpg). Images from IPA for the TR/RXR pathway for the ileum. Red shading indicates increased mRNA amounts of the respective gene during lactation, and green shading indicates decreased amounts of mRNA.

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Additional File 21:

RT-PCR primers (RT_PCR_primers.doc). Primer sequences for all genes analyzed by RT-PCR.

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