SuperScript en zyme was heat inactivated and the template RNA was then degraded upon incubation Vismodegib dosing with 5 units of RNaseH, for 30 min at 37 C. Quantitative Real Ttime PCR The experiments were carried out according to the MIQE guidelines. The first step for the primer se lections was to select from already published data a set of genes of interest differentially regulated during osteo genesis. The primer sequences were then se lected from a validated bank of oligos previously tested and approved for qRT PCR, the PrimerBank. The primer concentration was then optimized for each gene using a cDNA pool from different periods of time of treat ment with BMP2, adopting the lowest primer concentra tion for each condition that did not interfere with the amplification curve inclination, in order to avoid non specific results derived from primer dimers.

The qRT PCR reaction was carried out using 6 ul the SYBR Green Dye, 3 ul of 30 times di luted cDNA and 3 ul of a mix containing both the forward and the reverse primers, and incubated under the following conditions, 2 min at 50 C, 10 min at 95 C, followed by 40 cycles of 15 seconds at 95 C and 60 C for 1 min. The data were collected and analyzed using the 7300 System Software. The quality control of each reaction was achieved through a cycle of dissociation, in order to exclude possible cross contaminations or the presence of dimers. To confirm the differential expression for each gene, the GeneAmp 5700 software was used, and the threshold was set to 0. 1. The data was exported and interpreted using the qBASEPLUS2.

The first step was to use the Genorm tool, a very popular algorithm that finds the stablest reference genes from a set of tested candidate reference genes in a given experi mental condition, in this case, GAPDH, HMBS and HPRT. From this, a gene expression normalization factor was calculated for each sample, based on the geometric mean of a user defined number of the reference genes. After analysis, the data was exported and the graphic pic tures and statistical analysis were performed using the GraphPad Prism 5 software. The data presented in this work are representative of 3 independent experiments, performed in duplicates, and were analised through a one way Anova followed by a post test of Tukey with p 0. 005. CTCF is a highly conserved and ubiquitous protein that has widespread functions in transcription regulation and chromatin architecture.

It acts as a silencing and activat ing transcriptional factor, a chromatin insulator and a mediator of chromatin looping, and is GSK-3 essential for life. Binding of CTCF to DNA is achieved primarily through its 11 zinc finger domain, which also facilitates protein protein interactions. CTCFL or BORIS, is a paralo gue of CTCF. BORIS has almost identical 11 zinc finger domains to CTCF, and the proteins are thought to have evolved during vertebrate development from a gene duplication event.