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.
