Some histone genes are specifically expressed in hESCs and are preferentially associated with pluripotency. (a) The relative expression (normalized read densities) of all histone genes in both H9 and HeLa cells. Note that a number of histone genes showed significantly higher expression in H9 cells compared to that in HeLa cells, while a few showed a HeLa cell-specific expression pattern. (b) Validation of cell-specific histone mRNA expression by semi-quantitative RT-PCR using RNAs prepared according to their 3' end status. (c) Pluripotency-associated histone gene expression. Total RNAs were collected from different cell lines or cells treated under differentiation or reprogramming conditions, and were then treated with DNaseI before being subjected to semi-quantitative RT-PCR analysis. Some histone mRNAs (hist1h1d, hist1h2b, hist1h3i, and hist1h3j) were found to be preferentially expressed in undifferentiated stem cells and in reprogrammed cells, but their expression rapidly decreased upon differentiation and was low prior to reprogramming. hcgβ is a marker for trophoblast differentiation; oct3/4 and lin28 are pluripotency markers; and actin was used as a loading control.