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To understand the answer to this question, you have to understand what quantitative real time PCR (qRT-PCR) is measuring. Typically qRT-PCR is used to determine changes in gene expression at the mRNA level between different samples. First RNA is extracted from a sample, and reverse transcriptase is used to convert all the RNA to DNA, which is termed complimentary DNA or cDNA. If you start out with a certain amount of mRNA in each sample, the ensuing cDNA will be proportional to it, and will now be compatible with the polymerase chain reaction.

Once you have cDNA, using DNA primers specific to the gene you want to measure, you amplify your gene of interest. Assuming 100% efficiency, each cycle will double the amount of cDNA from the previous cycle. A qRT-PCR machine measures the relative level of cDNA after each cycle by reading the fluorescence from a probe that binds cDNA, and only fluoresces when bound. (There are also other probes but that's another discussion.)

At the end of the reaction, you will have exponential J shaped curves corresponding to each sample that flatten out at the top. If one sample had less of the gene you are measuring, its curve will be farther to the right, indicating that it took longer for the cDNA to amplify to any level (or fluorescence value) on the Y axis. If you draw a horizontal line through the curves in their exponential region, and then solve for the cycle number it took to get to the point where the curve and your line intersect, you will get the crossing point.

The significance of this is that now you have some value by which to compare samples with. If one samples crossing point is 20 and another's is 22, the sample with a crossing point of 20 had 4 times as much cDNA to begin with of the target gene (2^2), or generally 2^dcp (difference in crossing points) gives the relative difference in starting cDNA.

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Q: What is the crossing point in real time PCR?
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