Overexpression vector for M1AP with a doxycycline-inducible vector confirmed that M1AP induced high MYC expression by real-time quantitative PCR

A pTRIPZ-<em>M1AP</em>-<em>GFP,</em> doxycycline-inducible lentiviral vector, was induced HEK293T cells and 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). <em>Beta-ACTIN</em> 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: <em>MYC:</em> 5′-CGACTCTGAGGAGGAACAAGAA-3′ (forward) and 5′-CAGCAGAAGGTGATCCAGACT-3′ (reverse), <i>β<em>-ACTIN</em></i>: 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.

本研究采用强力霉素诱导型慢病毒载体pTRIPZ-M1AP-GFP转染HEK293T细胞并诱导靶基因表达，随后参照试剂盒操作指南，使用RNeasy® Mini试剂盒（Qiagen公司）提取总RNA。以提取的总RNA为模板，借助ReverTra Ace® qPCR RT Master Mix（Toyobo公司）合成互补脱氧核糖核酸（cDNA）。以β-ACTIN作为内参基因。采用ABI Prism 7900HT实时荧光定量PCR系统（Applied Biosystems公司），搭配SYBR Mix完成定量PCR（qPCR）分析以检测RNA表达水平。本实验所用基因表达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比对，分析靶基因的信使核糖核酸（mRNA）表达水平。