Quantifications of <i>91H and H19</i> ectopic RNAs and endogenous <i>Igf2</i> RNA levels in transfected <i>H19</i> KO myoblast cell lines.

<p>(A) The <i>91H/H19</i> insert (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g001" target="_blank">Figure 1</a>) have been co-transfected with a hygromicin-resistance plasmid into <i>H19</i> KO myoblast cells. Ectopic <i>91H</i>, <i>H19</i> and endogenous <i>Igf2</i> mRNA (Igf2mRNA PCR amplicon) levels were determined by RT-qPCR in undifferentiated <i>H19</i> KO myoblasts untransfected or transfected (whole hygromycin-resistant transfected cell population) with the <i>91H/H19</i> insert. Results were normalized to <i>Gapdh</i> expression levels. (B) Comparison between the <i>H19</i> and <i>91H</i> ectopic RNA levels. RT-qPCR quantifications were performed in undifferentiated (ND) or differentiated (D). The whole hygromycin-resistant transfected cells (“whole”) and 6 isolated clones (left panel), as well as C2C12 myoblasts (right panel), were analysed. The ectopic <i>H19</i> RNA expression levels (open bars) were assessed by using qPCR primers located at <i>H19</i> exon-exon junctions (<i>H19</i> RNA PCR amplicon). The ectopic <i>91H</i> RNA levels (black bars) were quantified with the mC’ and mI1-mI3 PCR amplicons. Error bars correspond to s.e.m. of quantifications obtained with mC’ and the mean of mI PCR amplicons. Detailed data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923.s005" target="_blank">figure S5</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#s4" target="_blank">Materials & Methods</a> section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923.s008" target="_blank">Table S1</a>). Please note that the other PCR amplicons shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g001" target="_blank">Figure 1</a> also target the endogenous truncated <i>91H</i> RNA produced from the endogenous locus and therefore they could not be used to quantify ectopic transcripts. Sample names and transgene copy numbers are indicated below the histogram.