Thread by @MackayIM

If you're baking a cake, maybe one you haven't tried before, do you set the oven at a temperature, cook it for exactly xyz minutes, then turn the oven off, pull the cake out and eat it? Or do you leave the oven on while you check the cake is baked, and if it isn't leave it in?

In PCR'land we don't take our tubes out & test them, but we "set and forget" to a MAX baking time (=total number of cycles; at 40-50). We come back at the end & see the results. Virus positive samples show up *before* the run is completely finished (15-30 cycles, with some later)

In the real-world RT-PCR result below you can see a bunch of positive curves for flu (the negatives are the flat lines underneath the red threshold).
Doesn't matter what the final cycle number is (how long the oven was on), just that it allows all the ingredients to be cooked.

Leaving the cake analogy now (because it would just get confusing).

Where the curve for each sample crosses that red threshold 👆is called the "threshold cycle", or Ct.
You can see that the Cts are very different in cycle number from the final cycles (the endpoint of the PCR).

In fact, every patient sample in that real-world example👆 has a slightly different Ct, just reflecting that every person has a slightly different amount of virus in their sample (affected by lots of things); sometimes not so slight!

Some other info and a stylised PCR curve showing the endpoint (starred; what most non-users confuse with Ct when talking about cycles)

Happy to answer real questions.

Resources...
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https://twitter.com/MackayIM/status/1331440271716827137?s=20

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https://twitter.com/MackayIM/status/1345203080732635136?s=20

🧬virologydownunder.com/putting-pcr-in…