Introduction
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.
