MYC-Luciferase Reporter Assay and q-PCR for MYC at the same timing with Luc-assay

A luciferase reporter harboring -3.1 kb of the MYC promoter region was integrated into a pGL4 vector (Promega). A pT3.5-CAG-M1AP was generated with pENTR221-M1AP and pT3.5-CAG-DEST (Kurata M et al., PLoS One, 2018, PMID:30222773) using Gateway® LR clonase. The luciferase reporter vectors, the pT3.5-CAG-M1AP vector, and the pRL Renilla luciferase reporter vector were co-transfected in the HEK 293T cells. The samples were harvested 48 and 72 hours after the transfection. Each assay was biologically triplicated and repeated. Luciferase activities were measured using the dual-luciferase reporter assay system (Promega) and Lumat LB9507 (Perkin Elmer). RNA was harvested at the same time points and the expression of MYC mRNA was analyzed by qPCR. RNA was isolated using an RNeasyR Mini Kit (Qiagen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was generated from RNA with ReverTra AceR qPCR RT Master Mix (Toyobo). Beta-ACTIN was used as an endogenous control. Using the ABI Prism 7900HT (Applied Biosystems), quantitative PCR (qPCR) analysis was performed to quantify the RNA level using SYBR Mix. The sequences for the PCR primers used in gene expression were as follows: MYC: 5′-CGACTCTGAGGAGGAACAAGAA-3′ (forward) and 5′-CAGCAGAAGGTGATCCAGACT-3′ (reverse), β-ACTIN: 5′-CACAGAGCCTCGCCTTTGCC-3′ (forward) and 5′-CACAGAGCCTCGCCTTTGCC-3′ (reverse). The mRNA level of the targeted gene was analyzed by comparison with the standard calibration curve.