Regulasi Promoter Thymidine Kinase oleh Sp1 dan NF-kB: Potensi Misinterpretasi Hasil Uji Lusiferase

Abstract

he luciferase assay is widely used in molecular biology laboratories to determine the effects of transcription factors on target gene expression levels. The results of luciferase assays are highly dependent on the expression of the control plasmid. Therefore, control plasmids in dual-luciferase assays are designed to exhibit stable expression that is not affected by experimental treatments. However, this assumption has not been systematically tested in the context of activation by Sp1 and NF-κB. The aim of this study was to evaluate the effects of Sp1 and NF-κB on the activity of the commercial control plasmid pRL-TK (GenBank AF025846; Promega). The results showed that co-transfection of Sp1 or RelA NF-κB (250 ng) with pRL-TK (25 ng) induced Rluc expression by 95-fold and 50-fold, respectively. Sequence analysis revealed that this promoter contains fourteen Sp1-binding sites and one RelA-binding site. These findings indicate that the use of pRL-TK or other control plasmids containing the TK promoter may lead to biased data interpretation or errors in normalization. In addition, co-transfection of Sp1 or RelA NF-κB (250 ng) with pRL-Null (25 ng), which does not contain the TK promoter, also increased Rluc expression by 10-fold and 4-fold, respectively. By increasing the amounts of Sp1 and RelA NF-κB to 0.5 µg and decreasing the amount of transfected pRL-Null to 12.5 ng, activation of pRL-Null by these two transcription factors could be minimized. It can be concluded that plasmids containing the TK promoter should not be used as expression controls in studies involving Sp1 or NF-κB, unless accompanied by preliminary testing to assess potential non-specific activation.