The University of Surrey researchers are studying how cells respond to different drugs by measuring the expression of genes such as those encoding drug metabolising enzymes, nuclear transport factors and transcription factors. The aim of the work is to gain better understanding of the mechanisms by which drugs are absorbed and metabolised by the body, using cultured cells (in particular liver, gut, neural and muscle) as a model. In a new project, they are also looking at the circadian profile of genes involved in the response to drugs in order to determine whether the time of day might affect drug metabolism and toxicity.
Once the cells have been exposed to compounds in culture, total RNA is extracted using the Machery-Nagel NucleoSpin RNA II Kit (available to UK scientists through Thermo Fisher Scientific, Epsom UK). This simple and rapid isolation procedure provides high-quality RNA suitable for quantitative PCR. This is followed by a reverse transcriptase step where target cDNA is amplified by qPCR using the ABsolute™ qPCR ROX Master Mix on an ABI 7000 Real Time PCR platform.
The team began using the ABsolute qPCR ROX Master Mix approximately five years ago, shortly after molecular biologist Dr. Kate Plant joined the department. “Initially, our decision to try another product was based on cost, but when we compared the ABsolute qPCR product with our previous mix, we also saw an improvement in sensitivity,” said Dr. Plant. “It was mainly for this reason that we decided to switch.”
“At around the same time,” Dr. Plant continues, “we also began to use the white Thermo Scientific ABgene ThermoFast® PCR detection plates. I had read on the Thermo Scientific/ABgene website that white plates would give better sensitivity for qPCR, and so I decided to compare them with natural plates. The white plates did indeed give us better sensitivity – results were coming up a cycle earlier compared to natural plates. There was one drawback, however; white plates made it more difficult to see which wells had been filled during set up.”
On the suggestion of her Thermo Scientific representative, Dr. Plant decided to try the new ABsolute Blue qPCR mix. She ran ABsolute Blue alongside her regular mix and found that it performed equally well. When she used ABsolute Blue in combination with the white PCR plates, however, the product really showed its value.
“ABsolute Blue made the set-up of reactions quicker and easier, as the mix was easier to see,” comments Dr. Plant. “We could see which wells had been filled and we could also see that all of the reagent had been dispelled from the pipette tips. It was much more visual and provided confirmation that set up and pipetting had been performed accurately, particularly useful for new students in the lab.”
“The white ThermoFast PCR detection plates and the ABsolute qPCR Master Mixes are a good combination,” concludes Kate, “giving excellent sensitivity. We typically take the standard curve to 10 copies and the Thermo Scientific QPCR products give us the performance we require”, concludes Dr. Plant.