With the introduction of fluorogenic probes and fluorometric thermal cyclers that enable the monitoring of amplification reactions in real time, the sensitivity and precision of gene expression analyses has improved dramatically. Utilizing these assays, often called quantitative or real-time RT-PCR, populations of mRNAs can now be analyzed from samples as small as a single cell. The sensitivity of these assays was significantly improved by the development of techniques such as laser-capture dissection for the isolation of individual cells from tissue slices.
However, improved sensitivity exposes problems inherent to extremely sensitive detection. In real-time PCR, the threshold cycle (the number of cycles of amplification required before fluorescence signals first become clearly visible) is linearly proportional to the logarithm of the number of template molecules initially present in the sample. However, when the sample possesses only a few target molecules, that relationship is subject to statistical uncertainties during the early stages of PCR. A sample containing only eight template molecules may behave like a sample possessing twenty-five molecules or like a sample containing only two molecules. Sometimes researchers can overcome this statistical limitation by analyzing the sample many times and then calculating the mean threshold cycle as a measure of target-copy number.
Since the number of mRNAs molecules in a cell varies from zero to a few thousand, with most species of mRNA being present in less than a hundred copies, when RNA species are amplified from a sample containing all of the RNA from a single cell, there is substantial uncertainty in the measured copy number. In order to overcome this problem, researchers developed more reliable ways to perform single-cell RT-PCR. One of these methods is based on digital PCR. In digital RT-PCR, a sample containing RNA from a single cell is divided into a large number of reaction wells, so that each well is likely to receive only a single template molecule. Fluorescent probes (such as molecular beacons or TaqMan probes) are utilized to light up the wells that contain amplified template molecules. The number of illuminated wells provides a direct measure of the number of target molecules in the sample.
Originally, digital PCR was performed in 96- or 384-well format and therefore had a dynamic range of 0–100 target molecules. However, the dynamic range of these assays has been improved by the introduction of assay platforms possessing thousands of individual wells (Figure 1). In one format, there are 3,000 wells etched into the surface of a glass slide, each with a volume of a few nanoliters. The sample is distributed among the wells, and real-time PCR is performed. In a related format, 1,200 chambers are created in a microfluidic device by the intersection of rows and columns of channels and valves.
In a third format, termed BEAMing (Beads, Emulsions, Amplification, and Magnetics), the sample is distributed into hundreds of thousands of microdroplets in a thermostable water-oil emulsion. Each microdroplet also contains a paramagnetic bead that binds to the amplified DNA and becomes labeled by fluorescent reporters associated with the amplified DNA. At the end of the amplification, the beads are separated from the emulsion and counted by a fluorescence-activated cell sorter. In the fourth format, the sample is diluted in a gel, and PCR is performed within the gel. Since the gel limits the diffusion of the amplicons during PCR, molecular colonies form at the positions of the original target molecules, and the number colonies indicates the number of targets that were in the original sample. Even more interesting formats are likely to follow.
Believing that the results obtained from exponential amplification of a few molecules will always suffer from statistical vagaries despite these innovations, other investigators have tried to do away with PCR altogether. A particularly attractive alternative is to perform in situ hybridization with several oligonucleotide probes against the mRNA target. Each of these probes is labeled with multiple fluorophores so that when they all bind to the same mRNA molecule at the same time, the target molecule appears as a fine fluorescent spot under a fluorescence microscope. All of the spots present in the cell can simply be counted, providing an accurate and integral value for the number of target mRNA molecules expressed in the cell.