Question

You are interested in determining the levels of specific mRNA in blood cells compared to brain cells. Using real-time PCR and equal number of cells in each sample, you find that a minimum level of detectable fluorescence is reached after 20 cycles of amplification starting with RNA from blood cells and in 10 cycles starting with RNA from brain cells. How would you interpret this result?

Answer 1

as we know that real-time PCR is used to determine the gene expression in particular samples (here we have brain and blood cells). hence we take equal number of cells and then we check how many cycles we need to see the amplification of DNA (The RNA which is first converted into a complementary DNA (cDNA)) which is detected in real time as the PCR is in progress. This is done by the use of a fluorescent reporter.

The fluorescent reporter signal strength is directly proportional to the number of amplified DNA molecules.

so as we need these number of cycles to get detectable amount of fluorescent -->

20 cycles of amplification starting with RNA from blood cells, and

10 cycles starting with RNA from brain cells.

here, we can see that more number of amplification cycles are needed in case of blood cells when compared to brain cells (and remember intially we had taken equal number of cells), this shows that the mRNA in blood cells is less as compared to brain cells. because in 10 cycles brain cell sample is showing the fluorescence which is seen in blood cell sample after 20 cycles. so we can say that for same fluorescence blood sample need more cycles.