Negative, or 'U-shaped' amplification curves

[superOK]

Hello

We have been having a strange problem with some of our Taqman assays, run on the Roche Lightcycler 480 using genomic DNA.

In some assays the baseline gradually decreases with each cycle, until the exponential phase where the signal picks up as normal. Sometimes the drop only gets to -2 or -3 units, but other times it can get to -20 before the exponential phase kicks in. As a guide, our reactions usually plateau in the region of 20-50 units.

It seems to mostly happen in the FAM channel, but has been seen in others (we use Yakima Yellow, FAM, ROX and Cy5).

Roche reckon it's not an instrument problem, but they didn't offer an alternative explanation.

Has anybody seen this problem, or does anybody have an explanation?

Thanks in advance!

[r.rosati]

This is already baseline-subtracted, I guess? It looks like the baseline extends way too far, until cycle 30 or so... Can you check the min and max offset on the "Background" button at the Cycle Range graph? Did you change the fist diplayed cycle there? Does the graph look exactly like that one you posted?

[r.rosati]

The Lightcycler 480 also has a second-derivative, "baseline-free" CT method, doesn't it? Does it give better data?

[mchlbrmn]

Proof an an anti-universe. The antimatter polymerase is sucking the DNA into the anti-universe, joining innumerable of my socks, keys, glasses, and one passport.

[r.rosati]

But the transfer is unstable Captain! The probe rematerializes in a few cycles!

Captain... is the anti-universe polymerase 6 feet tall, purple, and with lots of pseudopods?

[r.rosati]

Heheh sorry SuperOK, we changed the subject for a little bit. Any news?

[mchlbrmn]

But the transfer is unstable Captain! The probe rematerializes in a few cycles!

Captain... is the anti-universe polymerase 6 feet tall, purple, and with lots of pseudopods?

Your lab at a meeting?

[superOK]

Haha, thanks for the replies

r.rosati:

The data is baseline subtracted, using the second derivative method. However, the raw data looks the same (though starts at ~70 rather than 0).

I checked the min and max offset in background - and they were 1 and 5 respectively. The graph looks as it did above.

I'm stumped!

[relaxin]

No, Rob is day-dreaming about Star Trek.

[toxlab]

So, does this occur with higher expression targets you have run? Your curves dont come up until after 30 cycles...that may be nonconsequential, but worth comparing to other targets.
Also, is it possible this has something to do with probe/primer interactions?

my two cents...

[r.rosati]

The fact is that the reporter, once separated from the quencher, should fluoresce and keep doing it until the end of the run, unless as mchlbrmn joked you have an anti-polymerase. I really don't see a physical explanation for that curve - the only fluorophore that would lower its fluorescence over time is the quencher, if it's not dark.
So, it must be an artifact due to the way the data is processed. The first culprit, still, would be the baseline. Remember that the "offset" is relative - so "Min 1- Max 5" can also mean "cycles 21 to 25" if the initial point is set to cycle 20. If you have such dilute templates, move the values a bit and see how the curve changes.
How does the raw fluorescence curve look?
Do replicates behave similarly?

[superOK]

Hi

The raw data looks the same - albeit higher. When the 'negative' curve is really bad the initial background reading is also really high - this then drops to a low number when the curve levels out (all values are positive). When the baseline is set, the plot becomes negative, but doesn't change shape.

I had a theory that there may be something damaging the probes and releasing free fluorophore prior to the PCR. This would give a high background, and with each round of PCR the 'free' dye gets bleached, decreasing the signal. When all the free dye is quenched the levels flatten out (as in the negative amps on the graph shown). Any dye on intact probes is protected from photobleaching by the quencher, so the positive signals still work. Or that might be nonsense (if it was true, wouldn't the plateau actually decrease as well, since the free dye is bleached, or could it be that this would happen if you continued the PCR for long enough after the probe is exhausted?). Either way, I've ordered a new synthesis of the worst probe to see if it's any better.

This problem is worst with FAM. Yakima Yellow and Rox are affected, but only slightly. Cy5 works properly.

toxlab - I am using real time in genotyping rather than expression analysis. At higher gDNA concentrations I start to get inhibition. I will go back and check the theoretical primer/probe interactions to see if there is any pattern connecting the worst to the negative curves.

Thanks for all your input so far

[r.rosati]

The idea of initial probe release isn't bad... Nucleases maybe?
Could you share a printscreen of the raw fluorescence, best if on a plate with strong positives, weak positives and blanks?

...and this is why I was insisting on baseline:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665230/ (figure 1)
http://rtsf.msu.edu/Thresholds_and_Baselines.pdf (page 5)
It doesn't apply though, if you see it on the raw fluorescence data.

[ATeapoint]

Besides a screenshot of the raw data (which usually tells a lot), maybe you could also post a screenshot of the baseline setup?