The LightCycler™ System from Roche allows researchers to perform PCR reactions quickly while also giving them the ability to monitor the reaction in real-time. The thermal cycler (which holds up to 32 samples) uses heated air and glass capillaries with a high surface-to-volume ratio allowing each cycle of PCR to be completed in around 30 seconds – thus, a PCR run of 40 cycles can take just 30 minutes. The system, which comes with data analysis software, uses detection of fluorescence to monitor product amplification. It is set up for 2 formats, either using the dsDNA binding dye SYBR Green 1 or hybridization probes, which rely on FRET between the two probes when they hybridize in close proximity to each other on the target DNA. The addition of standards in the cycle run allows you to quantitate your samples by comparing them with the standards.
I have been using the LightCycler™ for RT-PCR and, when it is working well, it is great! Previously, I had used semi-quantitative RNase protections or radioactive RT-PCR to analyze gene expression levels in my samples. Whereas with those techniques it can take many hours (2 days in the case of RNase protection) to get any results, you can have results from the LightCycler™ in less than an hour once you have made your total RNA. I have not used the Lightcycler for its other suggested uses, such as detecting SNPs or analyzing multiple targets in the same reaction using differentially labeled hybridization probes.
As with all PCR you must be absolutely meticulous when setting up the reactions, as even very small errors in pipetting are amplified. The Roche protocol suggests adding 1ul of your template to each capillary and then aliquoting master mix into the capillaries. We now prefer to pre-mix the template and master mix aliquot before adding to the capillary as we find this gives more consistent results. Another minor irritation is the ease with which the glass capillaries break, so take care with those too.
The major disadvantage of this piece of equipment, however, is the cost of running it. For instance, if you need to spend time optimizing a new primer set, then the cost adds up. Since you must use 3-4 reactions on standards for each run on top of all of your samples this also adds to the cost. In addition, when the system is working smoothly it is so quick and simple, it can be very easy to get carried away and do run after run - some of which may not be absolutely necessary! Recently, however, other manufacturers have begun bringing out cheaper SYBR Green 1 kits suitable for use in the LightCycler™ , so hopefully prices will start to fall.
Fiona Wardle, Ph.D.
Post-Doctoral Fellow
Wellcome Trust/Cancer Research UK Institute
Cambridge, UK