EPSOM, England (October 22, 2009) – Thermo Fisher Scientific Inc., the world leader in serving science, is now assisting researchers at the University of Surrey, Guildford, UK as they study changes in gene expression within cultured human cells that are exposed to drugs and toxicants. Gene expression is measured by isolating total RNA and quantifying mRNA from target genes within the cells.
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.
For further information about the Thermo Scientific products for QPCR, visit www.thermo.com/gen.
Thermo Scientific is part of Thermo Fisher Scientific Inc., the world leader in serving science.
Photograph attached, caption: Dr Kate Plant and her team at the University of Surrey, Guildford, UK are using Thermo Scientific products as they study changes in gene expression within cultured human cells that are exposed to drugs and toxicants.
About Thermo Fisher Scientific
Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science, enabling our customers to make the world healthier, cleaner and safer. With 2008 revenues of $10.5 billion, we have approximately 34,000 employees and serve over 350,000 customers within pharmaceutical and biotech companies, hospitals and clinical diagnostic labs, universities, research institutions and government agencies, as well as environmental and industrial process control settings. Serving customers through two premier brands, Thermo Scientific and Fisher Scientific, we help solve analytical challenges from routine testing to complex research and discovery. Thermo Scientific offers customers a complete range of high-end analytical instruments as well as laboratory equipment, software, services, consumables and reagents to enable integrated laboratory workflow solutions. Fisher Scientific provides a complete portfolio of laboratory equipment, chemicals, supplies and services used in healthcare, scientific research, safety and education. Together, we offer the most convenient purchasing options to customers and continuously advance our technologies to accelerate the pace of scientific discovery, enhance value for customers and fuel growth for shareholders and employees alike. Visit www.thermofisher.com.
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.
For further information about the Thermo Scientific products for QPCR, visit www.thermo.com/gen.
Thermo Scientific is part of Thermo Fisher Scientific Inc., the world leader in serving science.
Photograph attached, caption: Dr Kate Plant and her team at the University of Surrey, Guildford, UK are using Thermo Scientific products as they study changes in gene expression within cultured human cells that are exposed to drugs and toxicants.
About Thermo Fisher Scientific
Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science, enabling our customers to make the world healthier, cleaner and safer. With 2008 revenues of $10.5 billion, we have approximately 34,000 employees and serve over 350,000 customers within pharmaceutical and biotech companies, hospitals and clinical diagnostic labs, universities, research institutions and government agencies, as well as environmental and industrial process control settings. Serving customers through two premier brands, Thermo Scientific and Fisher Scientific, we help solve analytical challenges from routine testing to complex research and discovery. Thermo Scientific offers customers a complete range of high-end analytical instruments as well as laboratory equipment, software, services, consumables and reagents to enable integrated laboratory workflow solutions. Fisher Scientific provides a complete portfolio of laboratory equipment, chemicals, supplies and services used in healthcare, scientific research, safety and education. Together, we offer the most convenient purchasing options to customers and continuously advance our technologies to accelerate the pace of scientific discovery, enhance value for customers and fuel growth for shareholders and employees alike. Visit www.thermofisher.com.
