In quantitative real time PCR experiment for the comparison of transcript levels

Question

In quantitative real time PCR experiment for the comparison of transcript levels of gene A in brain and liver, we observed that PCR products of mRNA in brain took 18 cycles to get to a certain abundance level. To get to the same threshold level, it took 24 PCR cycles for the liver sample Which sample had more copies of mRNA at the fist place. Can this experiment determine the exact copy number of the transcripts in both samples? Can this experiment determine the relative fold of copy number of the transcripts between these two samples? Can you calculate the relative transcript abundance of gene A between the two samples and express it in ratio (fold-change-linear scale) and log2 ratio (log-scale)? At threshold level Expression at threshold level in brain sample = Expression at threshold level in liver

Explanation / Answer

1. The brain sample had more copies of mRNA. This is because the cycle threshold is inversely proportional to the amount of nucleic acids present in the sample.

2. Ct values are used to correlate to the number of copies in the sample. However they cannot predict the exact copy numbers.

3. Yes. this experiment can predict the relative fold of copy number between these two samples.

4. relative quantification can be done using the formula: 2 Ct

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