-OFF human embryonic stem cells (hESCs) by detecting the expression of the
locus-derived non-coding RNAs (ncRNAs). (A) The DLK1-DIO3 imprinted locus, which is highly conserved between mice and humans, including clusters of maternally expressed functional ncRNAs, which are marked in red. The human homologs of the Gtl2 and Rian mice genes are MEG3 and MEG8, respectively. Lollipops with closed circles represent methylated CpG regions, and open circles represent unmethylated CpG regions. Mat, maternal chromosome; Pat, paternal chromosome. (B) The hESCs with high expression levels of imprinted long non-coding RNAs (lncRNAs) (MEG3 and MEG8) and of several imprinted microRNAs (miRNAs) from the DLK1-DIO3 locus (miR-127-3p, miR-154, miR-376c, miR-495, miR-494, and miR-496) were classified as MEG3-ON hESCs. The hESCs without detectable MEG3 expression accompanied by significant repression of other ncRNAs from the same locus were classified as MEG3-OFF hESCs. GAPDH was used as an internal control for mRNA expression analysis, and RNU48 was used as an internal control for miRNA expression analysis. The quantitation of lncRNA and miRNA expression was performed by using the 2−ΔΔCp method. Error bars represent the standard error of the mean generated from three biological repeats. **P <0.01 with respective MEG3-ON groups by Student’s t test. DLK1-DIO3, delta-like homolog 1 gene and the type III iodothyronine deiodinase gene; Gtl2, gene trap locus 2; IG-DMR, intergenic differentially methylated region; MEG3, maternally expressed gene 3; N.D., not detectable.