To validate the functional implication of the ASB16-AS1 gene in osteoblast, we performed gene overexpression experiment. Total cellular RNA was isolated from the human fetal osteoblastic 1.19 cell line (hFOB1.19, ATCC, Cat CRL-11372). Since the ASB16-AS1 transcription variant 2 uses an alternate splice site in the terminal exon compared to variant 1, the transcription variant 2 is shorter than variant 1. The same primers were used to amplify both the variants, only variant 2 was successfully subcloned into the pCEP4 vector. The primers for amplification of ASB16-AS1 were 5′-ggggtaccgtggcttcgcgactgcggaaggt-3′ (forward KpnI) and 5′- cgggatcctttttttttttttttttttatttttttttggtgcatactgtttaattttct-3′ (reverse BamHI). The procedure of cell culture was described previously [49]. Cells were seeded at 6 × 105 cells/well in 6-well plates. After 24 h, cells were transfected with Lipofectamine 3000, expression vector pCEP4-ASB16-AS1 using pCEP4 as a control. Real-time Polymerase Chain Reaction (RT-PCR) was used to detect the relative mRNA level of osteoblastic genes (BMP2 and ALPL) 48 h later after transfection. The primers for RT-PCR were shown in Table S1. Student’s t-test was applied to determine statistical significance.
