Question

I have a question about qPCR.

How to measure an mRNA level of a gene by qPCR. If I perform a realtive quantification qPCR, first I need to convert mRNA into DNA template by Reverse transcriptase. then I perform a qPCR reaction. For example, comparing with the reference gene, the drug treatment lead to a decrease in the expression of gene of interest. However, I think that can only determine the gene replication is inhibited because the pcr reaction is DNA replication , not DNA transcription. So, how can I know the difference in amount of mRNA in a cell?

Answer 1

qPCR is actually used to determine the amount of PCR product of DNA with respect of time.

Quantitative or semiquantitative (RTpcr) can be used to determine the expression in mRNA level.

First, you have to convert the mRNA into cDNA by a reverse transcriptase. Then, normal amplification of PCR product in which fluorescent molecule is used which fluoresces proportionally to the amount of double stranded DNA amplified. This binds to a particular sequence of the gene of interest.

so,for a particular time it will contain a few reaction cycles (round of pcr cycle) which is fixed.

Now if one cell express less the the amount of cDNA will be less and as the produced cDNA is the raw material, cell expressing more gene will give more DNA amplification after completion of a particular cycles.

thus you can differentiate between the expression of gene in normal and drug treated tissues.

Think,you have performed 35 cycles of pcr,

now sample which have more cDNA will acquire the threshold more quickly suppose at cycle 20 but the less cDNA containg sample will get threshold at cycles 25 .

thus you can differentiate by analyzing the graph of qPCR.