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.

将携带有MYC启动子区域-3.1 kb片段的荧光素酶报告基因整合至pGL4载体（Promega公司）。利用Gateway® LR重组酶，以pENTR221-M1AP与pT3.5-CAG-DEST载体为骨架（Kurata M等，PLoS One，2018，PMID:30222773），构建得到pT3.5-CAG-M1AP载体。将上述荧光素酶报告基因载体、pT3.5-CAG-M1AP载体与pRL海肾荧光素酶报告基因载体共转染至HEK 293T细胞中。分别于转染后48小时与72小时收集细胞样本。每项实验均设置3次生物学重复，并独立重复开展。采用双荧光素酶报告基因检测系统（Promega）与Lumat LB9507型发光检测仪（Perkin Elmer）检测荧光素酶活性。在相同时间点收集细胞总RNA，通过实时荧光定量PCR（qPCR）分析MYC mRNA的表达水平。依照厂商说明书，使用RNeasy® Mini试剂盒（Qiagen）提取总RNA。利用ReverTra Ace® qPCR RT Master Mix试剂盒（Toyobo）将总RNA反转录为互补脱氧核糖核酸（cDNA）。以β-肌动蛋白（Beta-ACTIN）作为内参基因。采用ABI Prism 7900HT型实时荧光定量PCR仪（Applied Biosystems），以SYBR Mix为反应试剂完成qPCR分析，以定量RNA表达水平。基因表达检测所用PCR引物序列如下：MYC上游引物：5′-CGACTCTGAGGAGGAACAAGAA-3′，下游引物：5′-CAGCAGAAGGTGATCCAGACT-3′；β-ACTIN上游引物：5′-CACAGAGCCTCGCCTTTGCC-3′，下游引物：5′-CACAGAGCCTCGCCTTTGCC-3′。通过与标准校准曲线对比，分析靶基因的mRNA表达水平。