Targeting rare cells
7 Feb 2007 by Evoluted New Media
Adult stem cells account for less than 1% of any tissue but they’re responsible for maintaining the integrity of tissues over a lifetime.
Adult stem cells account for less than 1% of any tissue but they’re responsible for maintaining the integrity of tissues over a lifetime.
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| EpiStem used the Eppendorf Mastercycler ep realplex to carry out gene expression profiling of potential therapeutic targets in untreated and chemotherapy treated mouse colonic crypts. |
PCR, developed in the 1980s by Kary Mullis, can amplify specific pieces of DNA more than a billion-fold using a DNA polymerase enzyme in conjunction with short, sequence-specific oligonucleotides which act as primers for the reaction. By exploiting heat-stable DNA polymerases it is possible to facilitate a chain reaction using a thermal cycler to provide rapid alternation of heating and cooling of the reaction tubes. Heating separates newly generated double-stranded DNA, while cooling allows the primers to anneal to this now single-stranded template DNA and the polymerase to initiate the next round of elongation, generating new complete strands of DNA.
Traditional PCR methods relied on detection of nucleic acids at the end-point of the reaction using imprecise methods such as gel electrophoresis to detect amplification products. Real-time chemistries combine DNA amplification and detection into a homogeneous assay in a single tube. Detection of the amplification product, based on changes in fluorescence, can now take place during the early, exponential phase of the reaction when the amount of specific double-stranded DNA increases two-fold with each PCR cycle, before the reaction plateau is reached.
It is this achievement which enables quantification of the starting amount of a specific DNA sequence in the sample before amplification, by monitoring how soon the signal from fluorescent reporter molecules reaches a threshold level. A wide variety of fluorescent chemistries are used, both sequence-specific probes and non-specific dyes such as SYBR Green which bind to double-stranded DNA. Multiplexing, whereby multiple targets are detected simultaneously in a single tube using probes labelled with different fluorophores, is also possible. Quantification may either involve comparison with a standard curve generated using a dilution series, or with a reference gene which acts as a control.
RNA can also be quantified using real-time PCR, by first converting it into complementary DNA using reverse transcriptase, an RNA-dependent DNA polymerase. With the right reaction conditions, the relative amount of a cDNA generated by reverse transcription will be proportional to the relative amount of its RNA template. The cDNA can then be amplified by PCR to determine even small changes in RNA levels and hence gene expression.
Discovering intestinal stem cell regulators
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.
Scientists at EpiStem are using Eppendorf’s Mastercycler ep realplex and epMotion 5070 to accelerate the discovery of these regulators. According to Dr Patricia Hurley, Senior Scientist in EpiStem’s Novel Therapies Division, “In order to study adult stem cells we need to be able to identify them and develop molecular methods capable of analysing very small numbers of cells. We use our proprietary EpiProfile technology in conjunction with automated real-time PCR to determine the messenger RNA expression profile in mouse intestinal crypts, where stem cells are located.”
Real-time PCR has a whole range of advantages over end-point detection, in addition to quantification. The method’s extreme sensitivity - detecting less than five copies of a target sequence - means that far smaller samples can be used, and the extremely wide dynamic range enables analysis of samples differing in target abundance by several orders of magnitude.
Real-time PCR is also faster than traditional PCR, with run times of well under an hour compared with several hours for traditional PCR. This eliminates the need for post-PCR processing, reducing the risk of cross-contamination in the lab as well as saving time. New reagent chemistries which do not require lengthy enzyme activation steps have also helped to shorten run times.
Automation of molecular biology methods is rapidly replacing classical manual protocols and another key advantage of real-time PCR is the potential to automate assays. Using robotic pipetting systems for assay set-up also improves accuracy and reproducibility. Sample replicate numbers can then be reduced and reaction volumes lowered, helping to cut costs and increase sample throughput.
Patricia Hurley explains the PCR setup, “Both instruments are small, really robust and it is easy to teach people to use them. The ep realplex gives us the flexibility to use any 96-well plates and any chemistries. We have been able to reduce reaction volumes to just 15 microlitres, rather than 25 with our previous method, with no loss of sensitivity. The instrument is highly reliable and running it eight hours a day provides cost-effective high-throughput screening.”
“We also use the ep Motion 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 samples equivalent in size to a clinical microbiopsy, the ability to work with minimum reaction volumes helps maximise the data we can obtain from our precious samples. We have already identified a promising group of targets from screening several thousand tissue samples, a task which would have been impossible without automated pipetting. These will then be taken forward into in vivo studies to test for possible therapeutic value. Real-time PCR provides much more accurate expression levels of individual genes, much more quickly compared with conventional PCR, and automation gives us the ability to analyse cDNA for many more genes, increasing the cost-effectiveness of assays.”
The modular design of Eppendorf’s 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 realplex optical module offers 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. Maximum flexibility is ensured by an open system concept and six different evaluation modules for results analysis.
As a method for quantifying specific RNA and DNA sequences, real-time PCR has become one of the most widely-used tools in molecular biology with applications in fields such as cancer biology, AIDS research, pathogen detection, virology, genotyping and analysis of mutations including single nucleotide polymorphisms (SNPs) which may predispose individuals to particular diseases.
Clinical diagnostic assays can be turned around significantly faster than by traditional culture methods. For example, real-time PCR was able to identify infection with the coronavirus responsible for the SARS outbreak in the first few days after disease onset when accurate diagnosis is most relevant for patient care. The capacity for high throughput of assays is vitally important in situations where large numbers of specimens need to be tested.
Real-time PCR is helping oncologists make more accurate assessments of a patient’s response to therapy, the presence and level of any minimal residual disease and the risk of recurrence of a malignancy. Disease-specific prognostic markers can be detected in leukaemia patients and impending clinical relapse after bone marrow transplant can be predicted.
Gene expression, in particular, is a rapidly widening area of investigation using real-time PCR. Further developments in instruments, chemistries, protocols and analysis software continue to provide the potential for even more accurate and efficient comparison of RNA levels.
By Geoff Simmons, Brand Marketing Specialist, Eppendorf UK
