Quantification of mRNA by RT-competitive-PCR and high performance liquid chromatography  

作　　者：Gary WILLIAMSON 

机构地区：[1]Nutrition and Consumer Science, Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK

出　　处：《Progress in Natural Science:Materials International》2002年第5期353-358,共6页自然科学进展·国际材料（英文版）

基　　金：Supported by the Biotechnology and Biological Sciences Research Council, UK

摘　　要：The use of RT-competitive-PCR with high performance liquid chromatography (HPLC) detection to quantify the absolute number of mRNA copies in mammalian cells is reported. As an example, the glutathione transferase (GST)-α mRNA in human hepatoma Hep G2 cells has been estimated. A PCR-generated internal standard was used as a competitor, co-amplified with the GST-α target sequence. The RT-competitive-PCR method was improved by designing target and competitor molecules which differed in only 30 base pairs. This allowed the two sequences to be co-amplified with the same efficiency. This improvement also facilitated a wider ratio to be used than previous methods (target:competitor ratio between 0.2 and 5). Products were baseline separated by HPLC using an ion-exchange column readily quantified at 260 nm. To validate the improved methodology, the effect of a known GST-α inducer, the anticancer drug oltipraz, was shown to induce GST-α mRNA up to 3-fold in Hep G2 cells. The RT-competitive PCR-HPLC method provides a reliable and sensitive way to quantify the amount of specific mRNA with 0.1 ng of total RNA.The use of RT-competitive-PCR with high performance liquid chromatography ( HPLC) detection to quantify the absolute number of mRNA copies in mammalian cells is reported. As an example, the glutathione transferase (GST)-α mRNA in human hep-atoma Hep G2 cells has been estimated. A PCR-generated internal standard was used as a competitor, co-amplified with the GST-α target sequence. The RT-competitive-PCR method was improved by designing target and competitor molecules which differed in only 30 base pairs. This allowed the two sequences to be co-amplified with the same efficiency. This improvement also facilitated a wider ratio to be used than previous methods (target:competitor ratio between 0.2 and 5). Products were baseline separated by HPLC using an ion-exchange column readily quantified at 260 nm. To validate the improved methodology, the effect of a known GST-a inducer, the anticancer drug oltipraz, was shown to induce GST-α mRNA up to 3-fold in Hep G2 cells. The RT-competitive PCR-HPLC method provides a reliable and sensitive way to quantify the amount of specific mRNA with 0.1 ng of total RNA.

关 键 词：RT-PCR  GLUTATHIONE transferase  oltipraz  HEP G2 cell  HPLC. 

分 类 号：Q522[生物学—生物化学]