Dual-luciferase reporter gene assays

The intron 2 region of TERT (including the rs2736100 flanking region) was amplified with human genomic DNA from healthy control individuals carrying either TERT rs2736100 TT genotype or rs2736100 GG genotype. Specific PCR primer pairs with the KpnI and XhoI restriction sites were showed in Supplementary Table 3. The PCR products were digested and ligated into an appropriately digested pGL3-Basic vector. The resultant TERT reporter gene plasmids were designated pTERT-T or pTERT-G, which were only different at the rs2736100 polymorphic site. Sanger sequencing of the insertions confirmed the orientation and integrity of the two constructs.

Both reporter gene constructs (pGL3-Basic, pTERT-T, or pTERT-G) and pRL-SV40 (Luciferase Assay System; Promega) were transfected into PTC cell line BCPAP cells or HEK293 cells. As previously described, dual luciferase activities were determined at 48 h after transfection²⁸. For each plasmid construct, three independent transfection experiments were performed, and each was done in triplicates.