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

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.

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

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.