How To Sure Reasonable Range Of Ct Values For qPCR Experiments

Ct value is the most important result presentation form of fluorescent quantitative PCR. It is used to calculate gene expression differences or gene copy number. So what is the Ct value of fluorescence quantification considered reasonable? How to ensure the effective range of Ct value?

What is Ct Value?

During the qPCR amplification process, the corresponding number of amplification cycles (Cycle Threshold) when the fluorescence signal of the amplified product reaches the set fluorescence threshold. C stands for Cycle and T stands for Threshold. Simply put, the Ct value is the number of cycles corresponding to when the initial template amplification reaches a certain amount of product in qPCR. The so-called "a certain amount of product" will be further explained later.

What does the Ct value do?

1.Relationship between exponential amplification, template amount and Ct value

Ideally, genes in qPCR are accumulated by exponential amplification after a certain number of cycles. The relationship between the number of amplification cycles and the amount of products is: Amplified product amount = initial template amount × (1+En) cycles number. However, the qPCR reaction is not always in an ideal situation. When the amount of amplified product reaches a "certain product amount", the number of cycles at this time is the Ct value, and it is in the exponential amplification period. The relationship between the Ct value and the amount of starting template: There is a linear relationship between the Ct value of the template and the logarithm of the starting copy number of the template. The higher the initial template concentration, the smaller the Ct value; the lower the initial template concentration, the larger the Ct value.

2.Amplification curve, fluorescence threshold and certain PCR product amount

The amount of qPCR amplification product is directly presented in the form of fluorescent signal, that is, the amplification curve. In the early stage of PCR, the amplification is under ideal conditions, the number of cycles is small, the product accumulation is small, and the level of fluorescence cannot be clearly distinguished from the fluorescence background. After that, the fluorescence increases and enters the exponential phase. The amount of PCR product can be detected at a certain point when the PCR reaction is just in the exponential phase, which can be used as "a certain amount of product", and the initial content of the template can be deduced from this. Therefore, the fluorescence signal intensity corresponding to a certain amount of product is the fluorescence threshold.

In the late stage of PCR, the amplification curve no longer shows exponential amplification, and enters the linear phase and plateau phase.

3.Reproducibility of Ct values

When the PCR cycle reaches the cycle number of the Ct value, it has just entered the true exponential amplification period. At this time, the small error has not been amplified, so the reproducibility of the Ct value is excellent, that is, the same template is amplified at different times or in different tubes at the same time. Amplification, the obtained Ct value is constant.

1.Amplification efficiency En

PCR amplification efficiency refers to the efficiency with which the polymerase converts the gene to be amplified into an amplicon. The amplification efficiency when one DNA molecule is transformed into two DNA molecules is 100%. Amplification efficiency is commonly expressed as En. In order to facilitate the analysis of subsequent articles, the factors that affect the amplification efficiency are briefly introduced.

2. Range of Ct values

Ct values range from 15-35. If the Ct value is less than 15, it is considered that the amplification is within the range of the baseline period and the fluorescence threshold has not been reached. Ideally, there is a linear relationship between the Ct value and the logarithm of the initial copy number of the template, that is, the standard curve. Through the standard curve, when the amplification efficiency is 100%, the calculated Ct value for quantifying the single copy number of the gene is around 35. If it is greater than 35, the initial copy number of the template is theoretically less than 1, which can be considered meaningless.

For different gene Ct ranges, due to the difference in gene copy number and amplification efficiency in the initial template amount, it is necessary to make a standard curve for the gene and calculate the linear detection range of the gene.

3.Influencing factors of Ct value

From the relationship between the number of amplification cycles and the amount of product: amount of amplified product = amount of initial template × (1+En) cycle number, it can be seen that under ideal conditions, the amount of initial template and En will have a negative impact on Ct value is affected. The difference in template quality or amplification efficiency will cause the Ct value to be too large or too small.

4.Ct value is too large or too small