Hey,

your curve looks absolutely normal! It might be easier to interprete the derivate (R') curve, which will give you one single peak at the temperature where the DNA denaturates.

Did you look at the data in derivative view?

Hey,

your curve looks absolutely normal! It might be easier to interprete the derivate (R') curve, which will give you one single peak at the temperature where the DNA denaturates.

I see cleary two products, but to view better the second derivate (SDM) is indeed necessary. When you enlarge the intervals at the X- axis the figure becomes more elongated. Many products melt at 81,5 oC (to be considerd as a SDM in the melting curve) this indicates 50% GC. Higher GC- melts at higher temperature. This might be the case at green/orange curves. This seems a different product

I agree: derivate (R') curves give more info. The indentation in the curve may be due to specific amplification. In that case you will observe two melting peaks in in derivative view. Try loading your amplicon in an agarose gel and check band specificity.

Hi there,

If one or more samples on a plate contains an unusually high level of fluorescent noise during the early cycles of PCR, the amplification curves of these samples may take on an irregular (typically sigmoidal) shape. Though rare, this effect can be seen when using the Auto Baseline analysis feature.

High fluorescent noise may be caused by one of two factors:

The assay itself may have an unusual amount of background signal, in which case all samples for a particular assay are likely to take on an irregular shape:

This happens more commonly with SYBR® Green than with TaqMan® assays, and is often corrected by switching chemistries. Otherwise, to correct for the problem during data analysis, you may try switching to Manual Baseline for the offending assay. For more details please visit:

http://www.appliedbiosystems.com/absite/us/en/home/support/tutorials/realtime-pcr-trouble-shooting-guide.html

As stated by others, a melting curve is almost impossible to interpret until it's been converted to the derivative. About the only things I can tell from the figure you provide is that the left-most (brown and yellow?) traces suggest a double melting peak and there are different products in different wells as indicated by the different Tms.

What do you mean by "notched"? (And you mention both amplification and melting but show only the melt.)

your data seem fine but I suggest you to look at the curves in derivative view so you can a better idea of both your amplification and melting curves.

I would suggest studying troubleshooting tips here:

http://www.slideshare.net/idtdna/troubleshooting-qpcr-what-are-my-amplification-curves-telling-me

Sure, it looks quite normal except some noise. I added the plot, just in case.

Thanks

Aren't there possibly two products plus primer dimers there? Are those curves for a single target gene?

If you are amplifying a single target, you definitely have non-specific products!

I suggest optimising the amplification condition, annealing temperature in particular.

And this is how the amplification plot looks like.

Actually in this reaction I run 5 different genes, but I did not like the shape of the curves, as for me they should be more smooth.

You mention you run 5 different genes on this plot. I would recommend improving your pipetting techniques to narrow the differences between your replicates. I believe this also caused the double peak in one of your genes plot. Best wishes.

I'm agree with you, the plateau region could by smooth. In my experience I would try to repeat the reaction if possible with a positive control, which you are sure that is correctly amplified. But before this, let check if the baseline value are set correctly. Moreover I usually analyze the plot with the dRn on the Y axis and I observe higher values.

The problem with your melting curve is due to this problem. I think that you could adjust the reaction maybe trying adding more DNA o RNA..it is an hypothesis..

Thanks a lot everybody, your suggestions were very helpful.

Makhsud

You've got split peaks and a few shoulders on some of them as well which suggests something is amiss unfortunately (which you couldn't see from the first melt curve - but is clear as day in the derivative).

It looks like your amp curve does not have any smoothing. It's essentially straight lines connecting the data points taken at each cycle. That's fine. When you see smooth curves (as on the Roche and BioRad CFX), there's some sort of smoothing algorithm applied to the visualized data.

But it looks like you have a lot of noise. As mentioned above, you should definitely attempt to optimize your amplification conditions. A good way to tell if you are getting efficient amplification is to amplify a dilution series and look at the slope of your standard curve. It's a good check on any new primer set.

If you continue to see noisy amplification, you could also switch to one of the brighter dyes (Promega's BRYT Green, or LTI's Eva Green) to improve your signal:noise ratio.

p.s. Your melt curve looks fine, and is always easiest studies as the derivative curve (as mentioned by other commenters). Again, the signal:noise ratio is high, even though you had fairly early amplification, suggesting either poor efficiency or low total fluorescence. Optimize primers and/or switch to one of the newer, brighter dyes.

Hi

Looking at your second figure of melting curve, I think there is some non-specific binding of primer other than target. It should look like the figure I have attached from one of my experiment. Also, you have figure out using different concentration of primer or your template. Hope this of help.

I think there is several problems in your qPCR:

If all your curves correspond to the same product, you should obtain the same peak (only one) (same melting temperature) for all your samples in derivative view. If there is some shifts between the peaks, so slightly different t°, it can be due to ionic strength differences ie pipetting errors (use low retention tips and very precise pipetor !!).

You probably have primers-dimers in the three left curves.

Some green curves clearly show two peaks !! So two products perhaps splicing variants ?? How were designed your primers ? As said above, a gel will give more details.

In fact I 'm not sure that you need ROX with the Applied Biosystems 7500.

Figure.

I like to see the SDM of the curves! Give much insight.

Did you observe the second product with a somewhat higher SDM? I saw it already in the first derivate curves. In my opnion are these not your desired amplicon but by-prodcuts. Did you run agarose electrophoresis to confirm the presence of by-product? Seeing your amplification curves I am almost sure of it. You see 4 curves appearing some Cq after the others. You did not show the log-plot, but I think they also have a different efiiciency. That also points to by-products, e.g. hetero (primer) dimers, selfamplification of primers due to internal loops.

You may need to check your analysis set up as well. When you did define, make sure choose the correct option relevant to your SYBR. If I understood correctly, in your case you should choose "ROX" option. But if there is no ROX in your SYBR, choose "NONE" option. Hope it helps.

I think your melting curve is normal. The notched shape of the melting curve is due to the numbers of domain. Domains are due to different GC content between adjacent sequence within the same amplicon (PCR product). I am using the high resolution melting curves analysis (hrMCA) method for genetic analysis of diseases and I have many experiences of different domain amplicons. In my case, we can use up to two domains and should change the primer pair when the domains are three and above for hrMCA analysis.

Hi,

Regarding Your question I think the problem of this kind of shapes may be due to cross amplificaions and could be a result of not fully optimized PCR reaction. Also try to make a agaroze gel and check if there are some nonspecific products.

I am of the view that you should try a dilution series with different primer concentrations. Also an AGEF could give you a clear view of your product for before you carry on with the RT PCR

HI

You get to like the following diagram. I think there is several problems at your design of work.

Thank you very much, everybody. Now I hope I know how to optimize the reaction conditions.

Makhsud