How to calculate fold change in real time pcr?
Answers
Answered by
0
There are two ways to analyze qPCR data: double delta Ct analysis and the relative standard curve method. With the assumption of equal primer efficiency, double delta Ct analysis caters to large amounts of DNA samples and a low number of genes to be tested. The standard curve method is more optimal if you have very few DNA samples but many genes to test.
Here, I explain the double delta Ct analysis (for a detailed explanation of double delta Ct analysis, read this paper).
Steps to conduct double delta Ct analysis
1. Take the average of the Ct values for the housekeeping gene and the gene being tested in the experimental and control conditions, returning 4 values. The 4 values are Gene being Tested Experimental (TE), Gene being Tested Control (TC), Housekeeping Gene Experimental (HE), and Housekeeping Gene Control (HC).
2. Calculate the differences between TE and HE (TE-HE) and TC and HC (TC-HC). These are your ΔCt values for the experimental (ΔCTE) and control (ΔCTC) conditions, respectively.
3. Then, calculate the difference between ΔCTE and ΔCTC (ΔCTE-ΔCTC) to arrive at the Double Delta Ct Value (ΔΔCt).
4. Since all calculations are in logarithm base 2, every time there is twice as much DNA, your Ct values decrease by 1 and will not halve. You need to calculate the value of 2^-ΔΔCt to get the expression fold change.
Here, I explain the double delta Ct analysis (for a detailed explanation of double delta Ct analysis, read this paper).
Steps to conduct double delta Ct analysis
1. Take the average of the Ct values for the housekeeping gene and the gene being tested in the experimental and control conditions, returning 4 values. The 4 values are Gene being Tested Experimental (TE), Gene being Tested Control (TC), Housekeeping Gene Experimental (HE), and Housekeeping Gene Control (HC).
2. Calculate the differences between TE and HE (TE-HE) and TC and HC (TC-HC). These are your ΔCt values for the experimental (ΔCTE) and control (ΔCTC) conditions, respectively.
3. Then, calculate the difference between ΔCTE and ΔCTC (ΔCTE-ΔCTC) to arrive at the Double Delta Ct Value (ΔΔCt).
4. Since all calculations are in logarithm base 2, every time there is twice as much DNA, your Ct values decrease by 1 and will not halve. You need to calculate the value of 2^-ΔΔCt to get the expression fold change.
Similar questions