Additional file 5: of Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering

IPA “Molecular and Cellular Functions” of AR-DE genes in riboflavin-rescued and riboflavin deficient chick embryos on embryonic day 15 (e15). A Microsoft Excel file containing nine worksheets of over-populated “Molecular and Cellular Functions” identified by IPA: “Fatty Acid Metabolism, Oxidation of Lipid, Carbohydrate (CHO) Metabolism, Catabolism of Protein, Metabolism of Amino Acids (AA), Protein Ubiquitination, Metabolism of Vitamins” and 29 AR-DE genes found in the chicken “Flavoproteome”. Each worksheet provides protein ID, gene symbol and gene expression as log2 ratio (Rf+/Rf-). (XLSX 35 kb

June 7, 2008 (Pages 2637-3226)

The primer sequences in qRT-PCR. (XLSX 10 kb

Transfection of synthetic miR-423-3p mimic negatively regulates Cox6a2, Ndufb7, and Ndufs5 as well as ATP level during C2C12 myotube induction.

<p>(A) Relative abundance of miRNA-423-3p by qPCR post miRNA-423-3p mimic transfection (n = 3). (B) 24 hours post-transfection, the cellular ATP level was lower (not significant) in the miR-423-3p mimic-transfected cells compared to the negative control at D0 and D4 and was significantly lower at D8. (C) Significant down-regulation of Cox6a2, Ndufb7, and Ndufs5 by qPCR 24 hours post-transfection. * and ** denotes p-values ≤ 0.05 and ≤ 0.01, respectively. All values are presented as mean ± SEM (n = 3).</p

Experimental scheme.

<p>(A) ATP measurement and expression profiling of microRNA and target mRNA. C2C12 myoblasts were maintained in growth medium (GM) for 2 days (80–90% confluence). Myogenic differentiation was then induced from D0 to D8 by switching to differentiated medium (DM). Cells were harvested for (1) intracellular ATP measurement (2) expression profiling of miRNA (Affymetrix GeneChip miRNA 3.0 Array) and target mRNAs (Qiagen RT2 Profiler PCR Array focusing on mitochondrial energy metabolism genes). (B) miR-423-3p overexpression by transfection of synthetic miR-423-3p mimic. C2C12 myoblasts were transfected with miR-423-3p mimic 24 hours prior to myotube induction (D -1), D3 and D7 post-induction and were then collected on D0, D4, and D8, respectively, for miRNA and target gene qPCR as well as ATP measurement.</p

Alteration of miRNA expression in C2C12 myoblasts post-myotube induction.

<p>(A) 15 down-regulated miRNAs that were negatively correlated with the ATP level (correlation coefficient, r = -0.67 to -0.83, P< 0.05). (B) 9 up-regulated miRNAs that were positively correlated with the ATP level (r = 0.67 to 0.79, P< 0.05).</p

Additional file 4: of Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering

Lists of “Analysis Ready” DE genes (AR-DE genes) assigned by IPA to “Canonical Pathways”. A Microsoft Excel file containing a five worksheets of the top “Canonical Pathways” identified by IPA in riboflavin-rescued and riboflavin deficient chick embryos on embryonic day 15 (e15). Five major canonical pathways identified by IPA among DE genes on embryonic day 15 (e15) were: “EIF2 Signaling, Acute-phase Signaling, LXR-RXR Activation, FXR-RXR Activation, Intrinsic Prothrombin Activation”, and “Blood Clotting” in the “Biological Function Category” of IPA. Each worksheet provides the gene symbol, Entrez gene name, gene expression as log2 ratio (Rf+/Rf-), and Ref-Seq protein ID or Entrez gene ID. (XLSX 27 kb

Confirmation of microRNA-microarray results by qPCR.

<p>miR-423-3p, miR-128-3p, and miR-301a-3p were selected for qPCR validation. Mean ± SEM (n = 4) of the log2 transformed microarray result (–•– on primary y-axis) and relative expression (2-ΔΔCt) derived from qPCR (—•—secondary y-axis) are overlaid. A correlation coefficient (r) and p-value are indicated.</p

List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.

<p>Data are shown as fold regulation levels compared to control group (D0) and normalized to mean of housekeeping genes (<i>Actb</i>, <i>B2m</i>, <i>Gapdh</i>, <i>Gusb</i>, and <i>Hsp90ab1</i>), then assessed for correlation with ATP levels.</p><p>List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.</p

Additional file 3: of Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering

Differentially-expressed (Adjusted P ≤ 0.05; FDR ≤ 0.05) genes identified in liver of e13 and e15 embryos. A Microsoft Excel file containing two worksheets. Work sheets “Riboflavin e13_396 DE genes” and “Riboflavin e15_1467 DE genes” list information about differentially expressed genes determined by microarray analysis. Each list provides the Roslin Institute Gallus gallus (RIGG) oligo ID, gene symbol, gene description, log2 fold change (Rf+/Rf-), average expression (Ave/Expr), t-statistic, P-value, P-value adjusted for multiple testing (adj.P ≤ 0.05), B-statistic from Limma software, and the Ref-Seq peptide ID for each gene (oligo). (XLSX 328 kb

miR-423-3p as a candidate miRNA for functional validation.

<p>(A) Conservation of miRNA-423-3p across species; Bos taurus (bta), Pan troglodytes (ptr), Mus musculus (mmu), Homo sapiens (hsa), Rattus norvegicus (rno), and Sus scrofa (ssc). The seed region (2–8 nt) is highlighted in a gray box and underlined. (B) Predicted targets of mmu-miR-423-3p in the 3′-UTR of Cox6a2, Ndufb7, and Ndufs5 (RNAhybrid). The prediction criteria include free energy ≥ -20 kcal/mol and allowing the G:U wobble base-pair and bulging nucleotides in the seed region. The seed match is underlined.</p