Hello Peers
I have been trying qPCR assay for AS1411 aptamer, a G-quadruplex aptamer. At the latest attempt, I got these weird curves that shifted down and left. The concentrations of the template were 2-fold diluted (A1 -> H1). Can anyone explain why it happened? The instrument is CFX duet from BioRad. I doubt the concentration of ROX but not sure about it. Any idea about it?
Sample prep for 1 rxn was:
* dNTP 10 mM 1µL
* Phusion polymerase 0.5 µL
* GC buffer 5x 10µL
* DMSO 1.5 µL
* Template 1 µL
* F primer 2.5 µL
* R primer 2.5 µL
* Water 29.5 µL
* ROX 2µM 0.5µL
* SYBR 10X 1µL
• Add-on (Dec 28th)
I forgot to say the concentrations of the template in the samples.
The range was 14.3 picomoles - 111.7 femtomoles per sample (50 µL), which is equivalent to the mass range 288.63 ng - 2.25 ng per sample. I assumed this range was low enough.
Also, I have doubted the same reason - too high concentrations. I attempted the lower concentration, but the same issue was observed.
The range was 27.9 femtomoles - 0.22 femtomoles per sample (50 µL), which is equivalent to the mass range 0.56 ng - 4.40 pg per sample.
The curves are attached.
It seems that your template concentration is way too high and the algorithm doing the baseline correction is confused/not working properly.
I guess it would help to dilute your samples by a factor of 1 to 10 million.
1 single molecule in your qPCR will give a Ct value of (about) 35 (more or less).
Ct values should not be lower than 15, which is 20 cycles earlier, corresponding to about 2^20 = 10^6 molecules.
1 fmol = 6 x 10^8 molecules, this should give a ct-value at about 6 cycles, 0.22 fmol -> about 8 cycles.
You lowest concentrations are still way too high.
However, it remains strange that the curves of the higher dilution look similar (except that they are much more noisy). They should show a similar noise and they should be shifted to the right (although not sufficiently much to get a robust identification of the exponential phase).
Go below 10^6 copies per reaction. You should get a proper amplification curve. If nothing changes, you may have a severe contamination (unlikely, because this would be really really drastic, but it would explain why diluting your sample does not shift the Ct values).
Is it possible to run a melting curve and an agarose gel or a PAGE to check that the correct product has been amplified?
Jochen Wilhelm
That makes sense a lot.
This might be a separate question tho, can the ROX concentration cause the same issue?
Not really, see here:
https://www.bio-rad.com/de-de/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye
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