Hello everyone,
I am having an issue with my qPCR assay, see pictures attached. I tried looking for anyone else getting graphs like this, but have not found an answer. The graphs are linear, for the Taqman primer sets but exponential when running SYBR green.
The primers and probe are 16S rRNA. They were obtained from a published paper, in which the graphs all look normal. I had two separate sets made when I initially started to get these results. One set has TAMRA as the quencher which is the published probe design, the other set has IDT's Zen IowaBlack double quenchers, of which I use extensively in lab. Both Taqman primers were run on a BioRad iQ5 and an Applied Biosystems 7500 Real time qPCR machine.
I also had the forward and reverse primers made separately, and ran them using SYBR green. The resulting graphs looked normal...so I have narrowed it down to the probe being the issue. I have tried decreasing the annealing temperature increasing the time, but that did not work either.
If anyone has any suggestions that would be great.
Thank you!!!
Hi Aesha,
you're right, it seems to be the probe. I think there are two possibilities: 1) the probe stock have some contaminant or inhibitor, or 2) there is a problem with probe annealing during PCR.
How do you prepare your qPCR mix? I prepare a mix of the primers and the probe (primers, probe, nuclease free H2O), and then mix it with the TaqMan master mix (master mix, mix of primers and probe, nuclease free H2O).
Try running an agarose gel to verify whether the right amplicon is obtained after qPCR.
Have you checked whether the probe sequence match that of your DNA template?
Best,
Hi, graph shape tell me that reaction effectiveness is insufficient. I am not sure, but it seems that fluorescence level is not high. For me, regarding TaqMan classical pair FAM (5' - reporter) and BHQ1 (3' - quencher) is the best.
You can try estimate reaction effectiveness with serial dillution followed by standard curve building. If you will see 90-99% or close to 2 (it depends on your soft) - ok, effectiveness is suitable. But if eff. is ok, the main reason of this view is quality of your probe, for example.
During my work I saw the same graphs. And if the reason is quality, please try to do the next for primer and probe mix before reaction:
+95oC-5' after that immediatly on ice and store before start preparation of reaction mix, or in the fridge (-20) for a 1-2 minutes, after that on ice or thermoblock (cooler) with 4-10 oC. Usually this step helps to increase general fluorescence level and improve graph.
I hope it usefull
Hi,
It looks like your PCR does not reach plateau conditions.
A few questions:
- Are your reaction conditions (supermix, Tm, cycling conditions, conc ....) the same?
- What is the Tm of your probe compared to your primers? Preferably your probe has a bit a higher Tm then your primers (5-8°). It allows the probe to sit ready on your amplicon already before the elongation and hydrolysis can take place. If the Tm is too low, the sensitivity might be compromised because not all target sites will be saturated with probe.
- What is the length of your amplicon? Preferably under 150 bp. Preferably the probe is close to one of the primers.
- Did you check for secondary structures etc. of the primers and probe?
.....
One tip: never just take primers and probes from publications. Always do a check for design parameters like Tm, secondary structures ... and definitely a BLASTor bisearch and do a correct wet-lab validation (including a dilution series, melt curve of the primers etc. on your samples).
Caroline Weydert is right regarding ......... One tip: never just take primers and probes from publications....It is very important advice.
Alejandro Piña Iturbe I used commercially available mastermixes. The primers and probe come in one tube all mixed together. I know I have contamination of E. coli DNA because my negatives all came up. We have narrowed that down to the Mastermixes, being contaminated since both have Taq polymerase made in E. coli.
Opening the tubes after amplifying is not an option as we want to minimize any potential amplicon contamination in our labs, especially with this amplicon. The sequence is present in the purified DNA, as the oligos are for E. coli 16S rRNA and the sample is also E. coli.
Thank you for taking the time to respond.
Andrei S. Babenka
Thank you for your all of your suggestions, I will try the modified PCR protocol, before my next run.
Caroline Weydert
Thank you for your suggestions.
As for your questions, the oligos do not form a secondary structure, the amplicon length is 118bp. The cycling parameters are the same, the amplification does plateau, see attached, it's the same data in log form, the graphs are just squished...if that makes sense.
Though, the Tm of the probe is 69C and the primers are 59C and 60C, and my annealing temp is 60C a little outside the norm, which maybe the issue though I wasn't expecting it to be so significant, but that just may be it.
Thank you for your suggestions, I will try a few more modifications with the cycling, to see if I can increase the fluorescences.
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