Thread by @MackayIM

What is a PCR cycle? 🧵
We sciencey types throw in words that we use for a specific purpose, but which also have other everyday meanings among most of the people on the planet.
We don't see that we do this ALL. THE. TIME.
"Cycle" is one of these words.

Here we're using "cycle" as it relates to the PCR = Polymerase Chain Reaction - a small, enzyme-driven cyclical DNA amplification reaction that we use to detect virus & do other sciency things (longer story)

When we do PCR to detect an otherwise undetectably teensy amount of DNA, we run 40 to 50 cycles of PCR. These 40 to 50 cycles (precise number varies by lab & kit) = the whole experiment (or "PCR run"), all of it.

Each of those 40-50 cycles is comprised of 2 or 3 (2 & 3 can be combined) *steps*.
1⃣A super hot step at 90-95'C
2⃣A cooler step at 50-60'C
3⃣A warmer step at 60-75'C
That would be 1 cycle.
Then we repeat that cycle, like a song on loop, 40-50 times.
That is a 40-50 cycle PCR.

This might be drawn on a graph of temperature versus time each step takes (plus time for the PCR machine to ramp up to & down from one temperature to the next), like this, for one cycle...

For example, you'll have heard "Canada runs all their PCRs at 45 cycles".
Well, yeah, lots of places & labs do.
That's not in any way important for the result.
You just run the experiment to the end-when the program & machine stops.
But results usually happen before that.

This cycle number is very different from saying "Bob's specimen had a Ct of 17 in the PCR".
That PCR still ran for 40-50 TOTAL cycles, it's just that the DNA was *detected* at 17 cycles. The lab result.
So you can also see that the result isn't affected at all by...

...how many TOTAL cycles the PCR ran for.
And the result came up well before the endpoint of the run.
This is the most common pattern for a PCR test - we run it for a few cycles more than the highest Ct values we expect to usually occur, based on our developmental experiments

Okay. This is a bonus thread for extra credit!
In the image I've made, you can see how the PCR in one graph as we see it in the lab (not all tests show it this way, unfortunately)
TOTAL number of cycles, or when the machine stops, is marked for each sample with a yellow star.

The point where each positive result occurs is marked with a yellow triangle; the Ct for that sample (we read the value from the bottom axis-pink arrow).
Some samples weren't positive because their fluorescence output stayed underneath our chosen threshold (red horizontal line)

I've also shaded two areas on that mock-up PCR result of positive & negative curves.
🟩Green is where we're happy that the result is strong - DNA was clearly detected.
🟥Red is where we'd like to maybe repeat the run, use a different test or collect a new sample & repeat.

We may even need to check the sequence of the virus if we start to see a trend of late positives, or weirdly shaped curves because it *might* indicate 1 or more mismatches between the primer/probe sequence & the sequence of the virus. Vigilance is ongoing.

For more on real-time PCR, check out...
virologydownunder.com/putting-pcr-in…