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| Figure 1. Ambion's QuantumRNA™ Technology in Multiplex Quantitative RT-PCR using 18S rRNA as an Internal Control. RT-PCR reactions on brain, embryo, liver, and spleen total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin, 18S rRNA primers and 18S rRNA Competimers. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance (B). Addition of Competimers (C) makes multiplex PCR possible, providing sample-to-sample relative quantitation. |
Ambion's QuantumRNA 18S Internal Standards contain 18S rRNA primers and competimers designed to amplify 18S rRNA in all eukaryotes. The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa. The Classic I and Classic II 18S Internal Standards can be used with any vertebrate RNA sample. All 18S Internal Standards work well in multiplex RT-PCR. These kits also include control RNA and an Instruction Manual detailing the series of experiments needed to make relative RT-PCR data significant. For those researchers who have validated ß-actin as an appropriate internal control for their system, the QuantumRNA ß-actin Internal Standards are available.
Competitive RT-PCR
Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence. The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis. The competitor is carefully quantitated and titrated into replicate RNA samples. Pilot experiments are used to find the range of competitor concentration where the experimental signal is most similar. Finally, the mass of product in the experimental samples is compared to the curve to determine the amount of a specific RNA present in the sample.
Some protocols use DNA competitors or random sequences for competitive RT-PCR. These competitors do not effectively control for variations in the RT reaction or for the amplification efficiency of the specific experimental sequence, as do RNA competitors. See The Accuracy of Competitive RT-PCR Depends on Using the Right Exogenous Standard for a further discussion on competitor choice and design.
Comparative RT-PCR
While exquisitely sensitive, both relative and competitive methods of qRT-PCR have drawbacks. Relative RT-PCR requires extensive optimization to ensure that the PCR is terminated when both the gene of interest and an internal control are in the exponential phase of amplification. Competitive RT-PCR requires that an exogenous "competitor" be synthesized for each target to be analyzed. However, comparative RT-PCR achieves the same level of sensitivity as these standard methods of qRT-PCR, with significantly less optimization. Target mRNAs from 2 samples are assayed simultaneously, each serving as a competitor for the other, making it possible to compare the relative abundance of target between samples. Comparative RT-PCR is ideal for analyzing target genes discovered by screening methods such as array analysis and differential display.
Tools for Any RT-PCR Technique
Whether you choose to perform real-time, relative, competitive, or comparative RT-PCR, Ambion offers products to simplify your RT-PCR experiments and make the data more quantitative. In addition to the specific products described above, Ambion offers SuperTaq™ Polymerase, M-MLV Reverse Transcriptase, and RNase-free PCR tubes. To prevent cross contamination during PCR experiments, Ambion also offers DNAZap™ DNA Degradation Solution and RNase-free barrier pipette tips.
For a comprehensive list of publications discussing practically every aspect of real-time RT-PCR please visit
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