The advent of real-time PCR and real-time reverse transcription PCR (real-time RT-PCR) has dramatically changed the field of measuring gene expression. Real-time PCR is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity (1
). Reactions are characterized by the point in time (or PCR cycle) where the target amplification is first detected. This value is usually referred to as cycle threshold (C<sub>t</sub>), the time at which fluorescence intensity is greater than background fluorescence. Consequently, the greater the quantity of target DNA in the starting material, the faster a significant increase in fluorescent signal will appear, yielding a lower C<sub>t</sub> (2
There are many benefits of using real-time PCR over other methods to quantify gene expression. It can produce quantitative data with an accurate dynamic range of 7 to 8 log orders of magnitude (3
) and does not require post-amplification manipulation. Real-time PCR assays are 10,000- to 100,000-fold more sensitive than RNase protection assays (4
), 1000-fold more sensitive than dot blot hybridization (5
), and can even detect a single copy of a specific transcript (6
). In addition, real-time PCR assays can reliably detect gene expression differences as small as 23% between samples (7
) and have lower coefficients of variation (cv; SYBR® Green at 14.2%; TaqMan® at 24%) than end point assays such as band densitometry (44.9%) and probe hybridization (45.1%) (8
). Real-time PCR can also discriminate between messenger RNAs (mRNAs) with almost identical sequences, requires much less RNA template than other methods of gene expression analysis, and can be relatively high-throughput given the proper equipment. The major disadvantage to real-time PCR is that it requires expensive equipment and reagents. In addition, due to its extremely high sensitivity, sound experimental design and an in-depth understanding of normalization techniques are imperative for accurate conclusions.
The general steps performed during a real-time PCR experiment, from RNA isolation to data analysis, are outlined in (Figure 1). This review is intended to provide an overview of the many facets of real-time PCR, highlighting PCR theory, quantification methods and models, data normalization, types of detection chemistry, and causes of variation.