You were interested in comparing metabolism-associated gene fatty acid synthase

Question

You were interested in comparing metabolism-associated gene fatty acid synthase (FAS) expression between fast growing pigs and slow growing pigs. You decided to use a Real-time PCR approach to measure liver FAS expression in these two lines of pigs. The housekeeping gene used in this study is GAPDH. Here is the data obtained from Real-time PCR: Ct(FAS) of a slow growing pig S is 35, Ct(GAPDH) of S is 25, Ct(FAS) of a fast growing pig F is 25, Ct(GAPDH) of F is 30. Estimate the fold difference of FAS expression between pigs S and F.

Explanation / Answer

CT value in real time pcr is the cycle threshold value.

It is defined as the number of cycles pcr was run before a real signal could be detected.

This value is obtained when a graph is plotted between number of cycles and signal .

It is the inverse of the amount of nucleic acid is in a sample.

Steps to obtain fold difference of FAS expression:-

1. To calculate CT

CT (slow) = CT of target gene (FAS) - CT of housekeeping gene (GADPH)

CT= 35-25 = 10

Similarly, CT (fast) = 25-30 =-5

2. To calculate (CT)

(CT) =CT of slow- Ct of fast

(CT) = 10- (-5) = 15

3. To calculate fold change

Fold change = 2^(-(CT)

Fold change= 2^ (-15) = 0.0000305175.

So, fold difference of FAS expression between pigs S and F is 0.0000305175.

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