Normalizing Target Genes to the Two Most Stable Reference Genes: <i>RNU1A/SNORD25</i>.

<p>Fold change for <i>miR-331-5p</i> and <i>miR-339-3p</i> between healthy endometrium (HE) and trophoblast (HT) were calculated using the geometric mean of <i>RNU1A/SNORD25</i> and the ddCt method. HE was compared to HT for each target gene independently by t-test. When the most stable pair of references genes predicted by geNorm and NormFinder algorithms was used as a normalizer, no significant differences in miRNA expression were observed between HE and HT. HE samples were arbitrarily set to a value of 1 + SEM (as a percentage of the variation among biological replicates), HT samples show the fold change above or below HE + SEM. Dark grey bars: healthy endometrium (n = 3), light grey bars: healthy trophoblast (n = 3).</p

Summary of Reference Gene Assessment.

<p>Summary of Reference Gene Assessment.</p

Importance of Reference Gene Stability on the Relative Quantification of Target Genes.

<p>Relative quantification of <i>miR-331-5p</i> (A) and <i>miR-339-3p</i> (B) with each reference gene in healthy endometrium and trophoblast. Differential expression of <i>miR-331-5p</i> and <i>miR-339-3p</i> is observed, even though microarray data [data not shown] indicated consistent expression between the tissues. Healthy endometrium (HE) was compared to healthy trophoblast (HT) independently for each reference gene by t-test. Data are shown as the mean + SEM, on a logarithmic scale. Dark grey bars: HE (n = 3), light grey bars: HT (n = 3). Significant differences (p<0.05) between HE and HT are demonstrated by asterisk (*) above the bars for the reference gene where the significant difference occurred.</p

Correlation of M Value and Stability Value.

<p>The correlation between the M value calculated by geNorm and Stability Value calculated by NormFinder was evaluated using the coefficient of determination (R²).</p

Real Time PCR Crossing Point Values in Pig Reproductive Tissues.

<p>Expression levels of all six candidate genes, shown as the mean crossing point (C_p) plus SEM for each tissue type. White bars: tissue collected from healthy conceptus attachment sites, black bars: tissue collected from arresting conceptus attachment sites, grey bars: non-pregnant endometrium. Means were compared by ANOVA and significant differences (p<0.05) are indicated by different letters above the bars of the graph. If bars have a letter in common, no significant difference exists. For all white and black bars, n = 3. For grey bars, n = 4. Endo: endometrium; NP: non-pregnant endometrium; Tropho: Trophoblast.</p

Candidate Reference Genes.

<p>Candidate Reference Genes.</p

Standard Curves and Melting Peaks of Candidate Genes.

<p>Standard curves were generated with a 10-fold dilution for each reference gene (A, D, G, J, M, P). Several dilutions were removed to optimize PCR efficiency (B, E, H, K, N, Q). Melting curve analysis revealed six single peaks, and different temperatures (C, F, I, L, O, R). Numbers on the graph indicate 10-fold dilutions remaining in the optimized standard curve.</p

Mouse estrous cycling.

<p>Mouse estrous cycling.</p

Vimentin staining of the ovaries and uterus.

<p>Vimentin staining of the ovaries and uterus.</p

Kidney, liver, ovary, and uterus histology.

<p>Kidney, liver, ovary, and uterus histology.</p