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

A pTRIPZ-M1AP-GFP, 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). 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.本实验对转染强力霉素诱导型慢病毒载体pTRIPZ-M1AP-GFP的HEK293T细胞进行诱导处理，随后依照制造商说明书，采用RNeasy® Mini试剂盒（Qiagen）提取总RNA。以Toyobo公司的ReverTra Ace® qPCR逆转录预混液，将总RNA反转录为互补脱氧核糖核酸（complementary DNA，cDNA）。以β-肌动蛋白（Beta-ACTIN）作为内参基因。使用Applied Biosystems公司的ABI Prism 7900HT型仪器，搭配SYBR Mix开展荧光定量聚合酶链反应（quantitative PCR，qPCR）分析，以定量检测RNA表达水平。本实验所用基因表达PCR引物序列如下：MYC基因上游引物：5′-CGACTCTGAGGAGGAACAAGAA-3′，下游引物：5′-CAGCAGAAGGTGATCCAGACT-3′；β-肌动蛋白（β-ACTIN）基因上游引物：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（messenger RNA，mRNA）表达水平。