RT-PCR: The Basics
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RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.
This article first discusses the advantages of real-time RT-PCR compared to end-point methods. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available. Subsequently, the “traditional” methods of quantitating gene expression by RT-PCR , i.e. end-point techniques, are presented.
Why Real-Time RT-PCR ?
Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale.
To truly appreciate the benefits of real-time PCR , a review of PCR fundamentals is necessary. At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made.
For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR . In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase.
Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad 107 -fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR -based quantitation technique.
Real-Time PCR Chemistries
Currently four different chemistries, TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons, Scorpions® and SYBR® Green (Molecular Probes), are available for real-time PCR . All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA.
TaqMan Probes
TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR , when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage
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