Northern blot analysis1/19/2024 All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. 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.Ĭurrently four different chemistries-Applied Biosystems™ TaqMan® and SYBR™ Green, Molecular Beacons, and Scorpions® chemstries-are available for real-time PCR. Data analysis, including standard curve generation and copy number calculation, is performed automatically. The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. 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. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made.įor 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). 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 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. To truly appreciate the benefits of real-time PCR, a review of PCR fundamentals is necessary. 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. 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.
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