Real-time PCR results can be analyzed using two main approaches: absolute quantification and relative quantification. Each method has its own advantages and applications, depending on the experimental goals.
Absolute quantification determines the exact number of target molecules in a sample by establishing a standard curve based on known concentrations. This method relies on the logarithmic relationship between the initial concentration of the DNA and the cycle threshold (Ct) value. To create a standard curve, serial dilutions of a known concentration are prepared. For example, starting with a stock solution, you can dilute it step by step by mixing 1 part sample with 9 parts dilution buffer to generate a series of standards (i, ii, iii, iv, v). Once the standard curve is established, the Ct value from the sample can be used to calculate the original copy number.
To calculate the copy number, first determine the sample concentration using the formula:
**Sample concentration (ng/µL) = OD260 × 40 × dilution factor**
Then, divide this by the molecular weight of the target gene, which is calculated as:
**Molecular weight = (number of base pairs × 324)**
Finally, multiply by 6 × 10¹ⴠto get the copy number per microliter.
Relative quantification, on the other hand, compares the expression levels of a target gene to a reference gene or a control sample. It does not require a standard curve but instead uses the difference in Ct values between samples. There are two common approaches: the double standard curve method and the double Ct method.
The double standard curve method involves creating separate standard curves for both the target gene and the reference gene. This allows for accurate comparison of their expression levels. However, this approach requires generating a new standard curve for each gene in every experiment, which can be time-consuming. It is often used in gene expression studies where the goal is to measure changes in expression under different conditions.
The double Ct method avoids the need for standard curves altogether. Instead, it calculates the relative expression based on the difference in Ct values between the target gene and the reference gene. This method assumes that both genes amplify with similar efficiency. If the amplification efficiencies are not consistent, the results may be less reliable. Despite this limitation, the double Ct method is widely used due to its simplicity and efficiency.
Both methods play important roles in real-time PCR analysis, and the choice depends on the specific research question and available resources. Whether you're studying gene expression, viral load, or genetic mutations, understanding these quantification techniques is essential for accurate and reproducible results.
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