Eppendorf real-time PCR helps target rare cells

EpiStem, a biotechnology company based in Manchester, UK, uses Eppendorf systems for automated real-time PCR assays, which are accelerating the discovery of key regulators for rare but vital stem cells in the small intestine.

The finger-like microvilli in the small intestine are responsible for absorbing all of the nutrients from the gut. Microvilli are replaced each week by a continuous production of cells that migrate like an escalator – from the crypts where stem cells are located to the tip of the microvilli where they’re sloughed off. Identifying the key regulators of this process would have enormous value for people suffering from cancer and inflammatory bowel disease.

Real-time PCR has a whole range of advantages over end-point detection methods: RNA quantification is possible, extreme sensitivity enables detection of less than five copies of a target sequence, dynamic range is extremely wide, and run times are well under an hour compared with several hours for traditional PCR.

Dr Patricia Hurley, Senior Scientist in EpiStem’s Novel Therapies Division, explains their choice of Eppendorf: ‘Our automated PCR set-up is based around the Mastercycler ep realplex and epMotion 5070 and 5075 liquid handling automated pipetting system. Both instruments are really robust and it is easy to teach people to use them. The ep realplex has enabled us to reduce our reaction volumes to just 15 microlitres with no loss of sensitivity. Using the epMotion automated pipetting systems for assay set-up has vastly improved our accuracy and reproducibility. We use the epMotion all day, every day, removing the user variation caused by hand-pipetting. It can pipette tiny volumes accurately and as we study really rare cell populations and very small clinical biopsy samples, the ability to work with minimum reaction volumes helps maximise the data we can obtain from our precious samples. We have already identified 300 promising targets from screening 5,000 or so, a task which would have been impossible without an automated system.’

According to Albrecht Wiener, Eppendorf UK Managing Director, ‘The modular design of our Mastercycler ep realplex offers researchers a choice of two thermomodules and two optical detection units, depending on their application requirements. The most advanced configuration of a 96-well silver block with the realplex⁴ optical module delivers high-speed heating and cooling rates and the capacity to undertake four-fold multiplexing assays. Fluorescent dyes are excited by 96 individual LEDs, which have a substantially longer lifespan than halogen lamps. Researchers have the flexibility to use any 96-well plates and any chemistries, while two different evaluation modules facilitate results analysis.’