竞争性RT-PCR法定量检测糖基化磷脂酰肌醇特异性磷脂酶D基因的表达

Competitive RT-PCR assay for quantification of GPI-PLD gene expression

目的 :建立一种定量检测糖基化磷脂酰肌醇特异性磷脂酶D(GPI PLD)基因表达水平的竞争性RT PCR方法 ,以研究GPI PLD与白血病等疾病的关系。方法 :首先用PCR定点诱变法构建GPI PLD的竞争性cDNA模板 ,该模板与靶cDNA具有相同的引物结合区 ,但在长度上要比靶cDNA少 2 0个碱基。把该竞争性cD NA模板在体外转录出的竞争性RNA作为RT PCR的内标准 ,与白血病细胞株K5 6 2细胞的总RNA在同一体系内进行逆转录和PCR扩增 ,二者的PCR产物因长度不同可经电泳区分开 ,然后对电泳图像进行光密度扫描 ,根据已知的竞争性RNA模板的量计算出GPI PLDmRNA的量 ,从而得到GPI PLD基因的绝对表达量。结果 :构建和制备了长度为 198bp的GPI PLD竞争性RNA模板 ,该模板与靶模板呈明显的竞争性RT PCR反应 ;测得K5 6 2细胞GPI PLD基因的表达水平为每纳克 (44 0± 2 0 )拷贝。结论 :成功地建立了一种定量检测GPI PLD基因表达水平的竞争性RT PCR方法 ,该方法具有灵敏、准确等优点。

Objective To establish a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to quantify the expression of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) gene. Methods A competitive GPI-PLD cDNA fragment was constructed by site-directed mutagenesis by PCR method . The competitive cDNA fragment had the same primer binding sites as the target cDNA , but was 20 bp shorter in length. The competitive cDNA was transcripted into the competitive RNA in vitro as the internal standard. Then the competitive RNA was reversely transcripted and amplified together with total RNA extracted from K562 cells. The two amplified cDNA fragments could be distinguished by 10% polyacrylamide gel electrophoresis. The concentration of cellular GPI-PLD mRNA was derived from the ratio between the intensities of the bands corresponding to the amplified products. Results A 198 bp GPI-PLD competitive RNA was constructed and prepared. The competitive RNA could compete well with the target RNA in the RT-PCR reaction. The expression of GPI-PLD gene in K562 cells was (440±20) copies∕ng. Conclusion A competitive RT-PCR assay for the quantification of GPI-PLD gene expression may be established successfully. This method is highly sensitive,specific,and accurate.