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Serial dilution calculator11/30/2023 ![]() ![]() An R 2 value >0.99 provides good confidence in correlating two values. If R 2 is 0, the value of X cannot be predicted from the value of Y (Figure 7B). ![]() When R 2 is 1, the value of Y (Ct) can be used to accurately predict the value of (Figure 7A). R 2 valueĪnother critical parameter to evaluating PCR efficiency is R 2, which is a statistical term that indicates how good one value is at predicting another. A PCR reaction with lower efficiency will have lower sensitivity. A slope of –3.3 ☑0% reflects an efficiency of 100% ☑0%. To accurately determine the efficiency of a PCR reaction, a 5-log dilution series must be performed. That means for 94% efficiency on a 5-log range, the assay would have a range of 88% to 100% efficiency. For the same number of dilutions or replicates on a 5-log range, the potential artifact is only ☘ %. Thus, even if the assay is 100% efficient, a range from 70 to 170% can be obtained when testing a dilution series of a single log, due to the standard deviation in one dilution. The reason for this suggested level of rigor is illustrated in Figure 6, which demonstrates the possible mathematical variation of slope or efficiency obtained when testing dilutions over 1 log vs. To properly evaluate PCR efficiency, a minimum of 3 replicates and a minimum of 5 logs of template concentration are necessary. In order to compare two reactions where a condition is changed (for example two different master mixes or two different instruments), the following parameters must be evaluated. Therefore, the absolute C t value comparison is only meaningful when comparing experiments using the same reaction conditions as defined above. However, this is not true if different instruments, reagents, primers and probes, or reaction volumes are involved in producing the two C ts. the other, could be valuable in concluding that there is less template in the first sample, assuming all other factors such as instruments, reagents, and assays are equal. Generally, an efficiency between 90 and 110% is considered acceptable. The PCR efficiency is dependent on the assay, the master mix performance, and sample quality. ![]() In this example, although the high-efficiency condition (the blue curve in Figure 5) gives a later C t at high concentrations, it results in better sensitivity at low target concentrations. In Figure 5, two samples (X and Y) amplified under low and high efficiency conditions show different C t values for the same target concentration. ![]() A dilution series amplified under low efficiency conditions could yield a standard curve with a different slope from one amplified under high efficiency conditions. The efficiency of a PCR reaction can also affect C t. Therefore, the C t values from PCR reactions run under different conditions or with different reagents cannot be compared directly. However, artifacts from the reaction mix or instrument that change the fluorescence measurements associated with the C t calculation will result in template-independent changes to the C t value. The C t value increases with a decreasing amount of template. The threshold must be set in the linear phase of the amplification plot in Figure 1C. The exponential phase in Figure 1B corresponds to the linear phase in Figure 1C. We will discuss the most common template-independent factors that can influence C t and describe how to evaluate the performance of a real-time PCR reaction.įigure 1, above, shows several parameters of the real-time reaction amplification plot. Many factors impact the absolute value of C t besides the concentration of the target. It is a relative measure of the concentration of target in the PCR reaction. C t (threshold cycle) is the intersection between an amplification curve and a threshold line ( Figure 1B). ![]()
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