Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.
There are some implications to this absolute count that should be understood prior to undertaking a digital experiment.
- Samples must be diluted to reach the digital range. Since we are now counting molecules in a small, dilute sample, instead of looking at a concentrated sample in a large volume, the mixing of your sample prior to making a dilution is critical. Each time you dilute a sample, you take a specific number of molecules from a tube and move them to another tube. No matter how well the sample is mixed, a different number of molecules will be removed from the tube each time you pipette from it. Good mixing will even this out, but they will never be exactly the same. This difference increases with each dilution step that you make. Because of this, you may want to consider running more than one sample from different dilutions of the same stock.
- The results returned measure the number of copies per µl in the solution that was put onto the chip. For this answer to have any meaning, you must have some idea of what you put on the chip. If you want to know copies per cell, then you will need to either count the cells before you start, or have some way of measuring the number of chromosomes in your sample, i.e. use a gene like RNAse P or Tert that has only one copy per chromosome in normal cells.
This is an exciting new technology, but it requires careful thought and planning to answer particular questions. Because of this, consultation is required prior to starting an experiment. Please contact us to make an appointment. You may find it helpful to read the appropriate section of this experiment design guide from Life Technologies prior to your appointment.