Pyrosequencing validation using whole genome-amplified and bisulfite modified mixtures of fully methylated and unmethylated DNAs.

<p>The percent of methylated DNA in each specimen analyzed is shown on the x-axis while the actual percent of methylation measured by pyrosequencing is shown on the y-axis. Error bars indicate the standard deviation for duplicate or triplicate measures.</p

Methylation at imprinted DMRs in the major umbilical cord blood fractions.

<p>(A) Representative Giemsa staining of peripheral blood monocytic cells (PBMCs) and polymorphonuclear cells (PMNs) from umbilical cord blood showing percent purity. (B) Methylation at the seven imprinted gene DMRs analyzed in this study did not significantly differ between blood fractions from up to 28 paired specimens analyzed based on paired t tests, except at the <i>MEG3-IG</i> DMR, for which the difference in mean methylation between fractions was 1.14%.</p

PCR and pyrosequencing primers (listed 5′ to 3′), genomic coordinates¹ and reaction conditions.

*<p>Biotin tagged primer.</p>1<p>Genomic coordinates based on UCSC Genome Browser, February 2009 release, GRCh37/hg19.</p

Differentially Methylated Regions of Imprinted Genes in Prenatal, Perinatal and Postnatal Human Tissues

<div><p>Epigenetic plasticity in relation to <em>in utero</em> exposures may mechanistically explain observed differences in the likelihood of developing common complex diseases including hypertension, diabetes and cardiovascular disease through the cumulative effects of subtle alterations in gene expression. Imprinted genes are essential mediators of growth and development and are characterized by differentially methylated regulatory regions (DMRs) that carry parental allele-specific methylation profiles. This theoretical 50% level of methylation provides a baseline from which endogenously- or exogenously-induced deviations in methylation can be detected. We quantified DNA methylation at imprinted gene DMRs in a large panel of human conceptal tissues, in matched buccal cell specimens collected at birth and at one year of age, and in the major cell fractions of umbilical cord blood to assess the stability of methylation at these regions. DNA methylation was measured using validated pyrosequencing assays at seven DMRs regulating the <em>IGF2/H19</em>, <em>DLK1/MEG3, MEST, NNAT</em> and <em>SGCE/PEG10</em> imprinted domains. DMR methylation did not significantly differ for the <em>H19</em>, <em>MEST</em> and <em>SGCE/PEG10</em> DMRs across all conceptal tissues analyzed (ANOVA p>0.10). Methylation differences at several DMRs were observed in tissues from brain (<em>IGF2</em> and <em>MEG3-IG</em> DMRs), liver (<em>IGF2</em> and <em>MEG3</em> DMRs) and placenta (both <em>DLK1/MEG3</em> DMRs and <em>NNAT</em> DMR). In most infants, methylation profiles in buccal cells at birth and at one year of age were comparable, as was methylation in the major cell fractions of umbilical cord blood. Several infants showed temporal deviations in methylation at multiple DMRs<b>.</b> Similarity of inter-individual and intra-individual methylation at some, but not all of the DMRs analyzed supports the possibility that methylation of these regions can serve as useful biosensors of exposure.</p> </div

Methylation of seven imprinted gene DMRs across a wide range of human tissues.

<p>The number of specimens analyzed is indicated within the parentheses after each tissue listed on the vertical axes. UMD, universally methylated DNA; error bars, SD across tissues; where only one tissue was analyzed, error bars represent the SD for replicate measures. ANOVA analysis (excluding UMD) p values are shown below each graph. Light grey bars designate those tissues showing deviation in methylation from the average; when removed, the p value becomes non-significant (i.e., p≥0.05).</p

Pyrosequencing validation using bisulfite modified methylated and unmethylated <i>IGF2</i> and <i>H19</i> DMR sequences in plasmids.

<p>Plasmids were quantified and mixed by pipetting to generate specimens containing 5% incremental increases in methylated DNA over the full range of possible methylation values (0% to 100%). Error bars, SD for duplicate measurements.</p

Intra-individual DMR methylation profiles in buccal cells over time.

<p>Matched buccal cell specimens from birth and one year of age (a subset of data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040924#pone-0040924-g006" target="_blank">Figure 6</a>), showing representative individuals with normal methylation profiles at both time points (panels A–D, infants 1–4) and those with normal methylation at birth that was abnormal for >1 DMR at one year of age (panels E–G, infants 5–8) as well as one individual with an abnormal methylation profile for 3 DMRs at birth that were normal by age one (infant 8). Paired t tests p values are shown for each infant. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040924#pone.0040924.s003" target="_blank">Table S3</a> for methylation values by DMR.</p

Deviation of methylation levels from 50% baseline across imprinted DMRs.

<p>Values shown represent the average of all methylation values obtained across all tissues analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040924#pone-0040924-g003" target="_blank">Figure 3</a>. The theoretical 50% level of methylation anticipated is represented as the baseline (0). Error bars, SD for all tissues analyzed.</p

The biological function of miR-133 during skeletal muscle development.

<p>MyoD modulate muscle proliferation by regulating expression of miR-133.</p

Hierarchical cluster analysis of the top 50 differentially expressed miRNAs (<i>P</i> < 0.05).

<p>Normalized log (base 2) data was hierarchically clustered by miRNA reads counts and is plotted as a heat map. LW1, LW2 and LW3 refer to the three biological replications of Large White pigs; MS1, MS2 and MS3 refer to the three biological replications of Meishan pigs.</p