1. In quantitative real time PCR experiment for the comparison of transcript lev
ID: 176444 • Letter: 1
Question
1. 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.
o Which sample had more copies of mRNA at the fist place.
o Can this experiment determine the exact copy number of the transcripts in both samples?
o Can this experiment determine the relative fold of copy number of the transcripts between these two samples?
o Can you calculate the relative transcript abundacne 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
The mRNA of brain has more copies of mRNA since it take less no of cycle to achieve certain level in comparison to the mRNA of liver. Yes, this experiment can determine the exact copies of the transcript in both samples because it is real time quantitative PCR. No, this experiment can not determine the relative fold of copy number of the transcript because there must be a reference mRNA for the determination of relative fold of copy number and since both the trancription occurs in different places of the body i.e. liver and brain, so it is not possible to relate each other.
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