Efficient extraction of RNA and analysis of gene expression in a long-term Taxus cell culture using real-time RT-PCR.

A simple, quick and efficient method for isolating total RNA from heavy browning cells was developed by adding polyvinylpyrrolidone, mercaptoethanol and 3 M NaAc during the process of the Trizol (a kind of a widely used RNA extraction buffer) method. High-quality total RNA was isolated and synthesized to cDNA. Transcript levels of four paclitaxel biosynthetic pathway genes: dxr, hmgr, ggpps and dbat were assayed by real-time RT-PCR. The results demonstrated that the transcript levels of these genes experienced a coincident descent in the past three years as well as a decreasing paclitaxel productivity. According to these results, the possible reason for the descending paclitaxel productivity during long-term Taxus media cv. Hicksii cell culture maybe due to a decreasing transcripts level of mass genes in close with a gross secondary metabolite level. Gene manipulation emphasized only on key enzyme genes in the paclitaxel biosynthesis pathway may not hamper the somaclonal variation trend of Taxus media cv. Hicksii cell culture.