Thread by @MackayIM

In the spirit of filling those with no PCR experience in. This is a lunchtime thread showing the progress of a reverse transcription polymerase chain reaction (RT-rPCR) "run".
The thing being amplified-the target or template-is a tiny amount of RNA I'd previously made

...in the lab ("in vitro transcribed RNA").
At this point there's too small an amount of RNA to view or use to get specific virus info from, so we put it through some chemistry & cycling temperature changes (the RT-PCR process) to copy it to levels we can "see".

RT-PCR isn't the only way to do this but its the topic here
So after the ivtRNA is added to a tiny tube (0.2ml) with enzyme, buffer, water, 2 primers and a probe, it's incubated at 50'C for a few minutes for RT enzyme to copy the RNA into complementary DNA (cDNA).
No signal yet

After that, the RT is heat-killed for a couple of minutes. We now should have enough cDNA for the PCR to "clone" it - make exact copies over & over again. This won't happen if there was no specific template there to begin with (unless you've designed yourself a rubbish test)

Each PCR cycle is made up of 2 or more steps. First, >92'C (temps vary with lab & kit). The 2nd may be combined with 3rd. If not (3-step) you drop the temp so that primers bind to their place on the target. 3rd, raise temp to >68'C (can vary) for 2nd enzyme to attach to primer..

and "grow" a new cloned strand. If you use two enzymes - you have an RNA-dependent DNA polymerase (RDDP or the RT) and a DNA dependent DNA polymerase (DDDP) that withstands the heat (another story). There are also single enzyme with both capabilities

Early on you don't see much of anything. What we're measuring in "real-time" is fluorescence emitted from the (most common home-brew or in-house" method) nuclease-destroyed probe. The DDDP does the destroying as it makes a new DNA copy. It meets the probe & eats it

Schematic of a dual-labelled nuclease ("TaqMan") probe binding between 2 primers. As it gets eaten by the DDDP, a fluorescent label (reporter, R)-previously dulled by a quencher (Q) label while they were bound close-is set free to emit fluorescence.
academic.oup.com/nar/article/30…

The reporter and quencher are chemically bound to a target specific "primer" (kind of-special chemistry) which is designed to "sit" between the primers so this process can happen. The probe doesn't bind if the specific target isn't present. Same with the primers.

By the way this is all happening in reaction volume that's 20 microlitres (millionths of a litre; can vary) and the whole process takes approximately 90min (guess what? That can vary). Every step so far can differ between labs. Certainly between diagnostic and research labs.

No target, no fluorescence = negative result (yes, even after 50 cycles). There's other stuff about sense & antisense strands which I won't go into (this isn't a @florian_krammer vaccine thread 😜)

So first the few cycles (measured along the bottom or x-axis) look like this. Boooorinng. Some of us like to check in on our important runs every few cycles. It's like clicking refresh on the concert ticket site!
On the left (y) axis we're have "normalised"👇 fluorescence.

Here, normalisation for each sample is done by taking the fluorescent data from the *background* of each sample-from up until just before amplification begins (2nd derivative) and averaging.
All data points for that sample are divided by that average.
Still not much to see👇

I'm getting a coffee (or setting up 27 more runs and writing these results up).

Hmm-what's that pink line doing? We're past cycle 30 now. Keep in mind that I already know this to be a positive template but its ivtRNA-not virus-so this isn't a run to detect an infection.
It was actually to see how repeatable the pipetting of the same RNA multiple times, was.

Something has happened! We can see that the fluorescence of a bunch of samples (each a pink line) has started to curve up. We *could* call this & go home now, but it's better to know what else is happening with other samples & controls & review the shape & height of each curve

Look at the exponential growth! Remind you of anything else?
Some spacing between the curves (replicates of my ivtRNA) there suggest a little variability in pipetting, but still pretty tight.

If we take the log of those data (this is done using the software that drives the PCR cycler) we can see a nice long exponential (linear bit that harshly tilts upward) phase - this is where we want to set or threshold so as to capture the actively "growing" fluorescence curve.

We don't want to include early background "noise" or late slowed reaction when we set the threshold. At the end, the exponential curve is falling toward a horizontal plateau. At this late point, our positive PCR reactions are overwhelmed by chemical by-products

..crowed by complete & incomplete products, hampered by misprimed things & just too little fresh material. It's tired.

This study of repeatability showed me that 10 replicates bunched between Cts of 31.44 & 32.51 (~1Ct spread). If I moved the threshold (I used my fave of 0.05 normalised fluorescent units; this can vary) ⬆or⬇, the Ct values change because they're defined by the crossing point

I'll also mention that the dual labelled probe can be labelled with different fluorescence-generating molecules. This one emitted in the green wavelengths labelled. But in the same run I had 10 replicates of another ivtRNA detected using a probe with an orange-emitting label

Oh & each set of 10 replicates also included two NTCs - no template controls-to which I added 5ul of water instead of the ivtRNA target. They didn't show any exponential curve which is what they're supposed to do. (line colours are my setting; unrelated to fluorescence colour)

Also, pipetting not as good on this (33.03-35.26; 2.23 spread 😞)!