Effect of Limiting Reagents
The early cycles of PCR are characterized by an exponential increase in target amplification. As reaction componentsbecome limiting, the rate of target amplification decreases until a plateau is reached and there is little or nonet increase in PCR product. The sensitive fluorescence detection of the 5700 and 7700 systems allows the thresholdcycle to be observed when PCR amplification is still in the exponential phase. This is the main reason why CT is amore reliable measure of starting copy number than an endpoint measurement of the amount of accumulated PCRproduct. During the exponential phase, none of the reaction components is limiting; as a result, CT values are veryreproducible for reactions with the same starting copy number. This leads to greatly improved precision in thequantitation of DNA and RNA. On the other hand, the amount of PCR product observed at the end of the reactionis very sensitive to slight variations in reaction components. This is because endpoint measurements are generallymade when the reaction is beyond the exponential phase and a slight difference in a limiting component can havea drastic effect on the final amount of product. For example, side reactions, like formation of primer dimers, canconsume reagents to different extents from tube to tube. Thus, it is possible for a sample with a higher starting copynumber to end up with less accumulated product than a sample with a lower starting copy number. The differencesbetween endpoint and real-time detection are graphically illustrated in Figure 4, which shows amplification of 96identical samples. The overall change in reporter signal, as measured at cycle 40, varies widely among the replicates.However, the amplification plots are remarkably similar between cycles 22 and 25, during which the CT valuesare determined.
Quantitative Competitive PCR
In order to compensate for problems with endpoint measurements, researchers have developed a variety of quantitativecompetitive PCR techniques.11–14 Typically, a competitor amplicon is constructed that contains the sameprimer binding sites and has the same amplification efficiency as the target, but is somehow distinguishable fromthe target. A common distinguishing characteristic is to make the target and competitor amplicons different sizesso that gel electrophoresis can be used to discriminate the two products. A known amount of competitor is spikedinto the sample, then the target and competitor are amplified in the same reaction. If the amplification efficiency oftarget and competitor are, in fact, identical, then the ratio of target to competitor will remain constant throughoutthe PCR process. Thus, by determining the ratio of target to competitor at the end of the reaction and knowing thestarting amount of competitor spiked in, the starting amount of target can be calculated. Competitive PCR has beenused successfully to quantitate DNA and RNA, but its dynamic range is limited to a target-to-competitor ratio ofabout 1:10 to 10:1. In fact, the best accuracy is obtained by finding the equivalence point at which the ratio oftarget to competitor is 1:1. To accomplish this, several dilutions must be tested in order to achieve a suitable ratioof target to competitor. Another drawback is the need to construct and characterize a different competitor for everytarget to be quantitated. In addition, careful validation studies must be performed in order to verify that the amplificationefficiencies of target and competitor are the same before quantitation of experimental samples can commence.Even a slight difference in efficiency severely compromises the accuracy of quantitation by competitive PCR. At theend of the reaction, competitive PCR requires accurate quantitation of target and competitor amplicons, whichusually entails laborious post-PCR processing steps.
Advantages of Real-Time
The development of competitive PCR was driven by a reliance on endpoint measurements. Determining CT valuesby following the real-time kinetics of PCR eliminates the need for a competitor to be co-amplified with the target.Quantitation can be performed by the more basic method of preparing a standard curve and determining unknownamount by comparison to the standard curve. Compared to endpoint measurements, the use of CT values alsoexpands the dynamic range of quantitation because data is collected for every cycle of PCR. A linear relationship between CT and initial DNA amount has been demonstrated for over five orders of magnitude, compared to the oneor two orders of magnitude typically observed with an endpoint assay. Real-time quantitation eliminates post-PCRprocessing of PCR products, which not only increases throughput and reduces the chances for carryover contamination,but also removes post-PCR processing as a potential source of error. Although not immune, CT values are less sensitive than endpoint values to the effects of PCR inhibitors, again, because measurements are from the exponential phase where reaction components are not limiting.
Quantitation of Cytokine Gene Expression
One application of the real-time quantitative capability of either the GeneAmp® 5700 Sequence DetectionSystem or the ABI PRISM® 7700 Sequence Detection System is to monitor how patterns of gene expression changein response to various stimuli. Figure 5 shows the relative quantitation results for three cytokine mRNAs in PBMCsthat have been stimulated with CD3/CD28 antibodies. Total RNA was prepared from untreated PBMCs and fromcells that had been exposed to CD3/CD28 antibodies for 2 hours, 6 hours, or overnight. The relative quantity ofIL-2, IL-4, and TNF-a mRNA in each sample was determined using either generic SYBR® Green I dye detection orgene-specific fluorogenic probes. Thermal cycling and detection of the real-time amplification plots were performedusing the 5700 and the 7700 Sequence Detection Systems. In order to perform these analyses, cDNAs prepared fromthe four total RNA samples were mixed with either TaqMan® Universal PCR Master Mix or a master mix formulatedfrom the components of the SYBR® Green PCR Core Reagents Kit. These master mixes contain all necessary reactioncomponents except primers and probes. Each cDNA mix was added to separate wells, each well containing the primerpair for amplification of one of the cytokine targets. Samples containing the primer pair for amplification of 18Sribosomal RNA were also prepared. For gene-specific detection, the wells also contained the appropriate fluorogenicprobe for the cytokine or ribosomal RNA target.
