The Reasonable Range of Ct Values in qPCR Experiments

Ct value is a crucial outcome representation in fluorescent quantitative PCR. It is used to calculate differences in gene expression levels or gene copy numbers. So, what range of Ct values can be considered reasonable in fluorescent quantitation? How can we ensure that the Ct values fall within an effective range? Today, let's allow Xiao Yi to answer this question for everyone.

What is Ct value?

In the qPCR amplification process, the Ct value refers to the number of amplification cycles (Cycle Threshold) at which the fluorescence signal of the amplified product reaches the set fluorescence threshold. "C" stands for Cycle, and "T" stands for Threshold. In simple terms, the Ct value is the number of cycles it takes for the starting template in qPCR to reach a certain amount of product, which will be further explained later.

What is the function of the Ct value?

1. Relationship between Exponential Amplification, Template Quantity, and Ct Value

In an ideal scenario, during qPCR, genes are exponentially amplified and accumulate over a certain number of cycles. The relationship between the number of amplification cycles and the amount of product can be expressed as: the amount of amplified product = initial template quantity×(1 + En)^number of cycles. However, qPCR reactions do not always occur under ideal conditions. When the amount of amplified product reaches a "certain product quantity," the number of cycles at this point is the Ct value, indicating the period of exponential amplification. The relationship between the Ct value and the initial template quantity is as follows: there is a linear relationship between the Ct value of a template and the logarithm of the initial copy number of that template. A higher concentration of initial template results in a lower Ct value, whereas a lower concentration of initial template leads to a higher Ct value.

2.Amplification Curve, Fluorescence Threshold, and a Certain Product Quantity

The quantity of qPCR amplification products is directly represented in the form of fluorescence signals, which is known as the amplification curve. In the early stages of PCR, when amplification is under ideal conditions and the number of cycles is low, the accumulation of products is minimal, and the level of fluorescence produced is not significantly distinguishable from the background fluorescence noise. Subsequently, the production of fluorescence enters the exponential phase. The amount of PCR product can be detected at a certain point when the reaction is just entering the exponential phase, which is referred to as the "certain product quantity." This can be used to infer the initial content of the template. Therefore, the fluorescence signal intensity corresponding to the certain product quantity is known as the fluorescence threshold.

In the later stages of PCR, the amplification curve no longer exhibits exponential amplification and enters the linear phase and the plateau phase.

3.The reproducibility of Ct values

When the PCR cycles reach the number of cycles at which the Ct value occurs, it is just entering the true exponential amplification phase. At this point, minor errors have not been amplified, so the reproducibility of Ct values is excellent. This means that whether the same template is amplified at different times or in different tubes at the same time, the obtained Ct values remain constant.

What is the range of Ct values?

1.Amplification efficiency En

PCR amplification efficiency refers to the efficiency with which the polymerase converts the target gene into amplification products. The amplification efficiency is 100% when one DNA molecule is converted into two DNA molecules. Amplification efficiency is commonly denoted as En. For the convenience of analysis in subsequent articles, here is a brief introduction to the factors that affect amplification efficiency.

2.The range of Ct values

The range of Ct values is 15-35. A Ct value less than 15 is considered to be within the baseline phase and has not reached the fluorescence threshold. Ideally, there is a linear relationship between the Ct value and the logarithm of the initial copy number of the template, which is known as the standard curve. Using the standard curve, when the amplification efficiency is 100%, the Ct value for quantifying a single copy of the gene is calculated to be around 35. If the Ct value is greater than 35, theoretically, the initial copy number of the template is less than 1, which can be considered statistically insignificant.

For different genes, the Ct value range, due to variations in the initial template amount of gene copies and amplification efficiency, requires the creation of a standard curve for that gene to calculate the linear detection range of the gene.

3. Factors affecting Ct values
As indicated by the relationship between the number of amplification cycles and the amount of product: Amplification product amount = Initial template amount × (1 + En)^number of cycles, it can be seen that under ideal conditions, the initial template amount and En will impact the Ct value. Differences in template quality or amplification efficiency can cause the Ct value to be too high or too low.

4. Ct values being too high or too low
Xiao Yi consulted the experts in our company's technical department and summarized the causes and solutions for the two common types of issues with Ct values to enlighten our readers.

This concludes the analysis of the abnormal Ct value causes for this issue. Next, Xiao Yi will recommend a range of cost-effective products to ensure your experimental data is more accurate and reliable. Come and pick your favorites!