Hi Nikoleta,

which machine are you using?  We've seen similar results sometimes with our old ABI7000 with the baseline calculation set to 'auto'.  If your machine is automatically removing the background signal, try doing it manually (i.e. I would suggest setting it from cycle 3 to cycle 12 for your second graph, although you can tell best when the amplification starts by not using a log-scale on the Y axis).  The strange flat signal at the top then might resolve itself.

This seems to occur when you have lots of sample / amplification occurring early and the software can't always cope, so try manual background removal first (if you're not already) and then try diluting your input template (DNA?  cDNA?) by at least 10x if not more - the results will still be accurate if your traces are coming up at later cycles.

Good luck

Hi Nikoleta,

which machine are you using?  We've seen similar results sometimes with our old ABI7000 with the baseline calculation set to 'auto'.  If your machine is automatically removing the background signal, try doing it manually (i.e. I would suggest setting it from cycle 3 to cycle 12 for your second graph, although you can tell best when the amplification starts by not using a log-scale on the Y axis).  The strange flat signal at the top then might resolve itself.

This seems to occur when you have lots of sample / amplification occurring early and the software can't always cope, so try manual background removal first (if you're not already) and then try diluting your input template (DNA?  cDNA?) by at least 10x if not more - the results will still be accurate if your traces are coming up at later cycles.

Good luck

Hi Nikoleta, I agree with Steven, I had same problems with the ABI7500. When I had a lot of cDNA, even after diluition, I see those sigmoidal curves, not only with SYBR but also with Taqman. The baseline was setting on automatic (in general cycle 3 to cycle 15) and the machine reads the real sample signal (that appear before the cycle15) as background.

For the replicates pay attention to your pipettes and for the diluition do not use small volumes. It seems impossible to have diffferent results using the samples tubes but it is not so strange if you do not follow some rules.

I also use life technologies troubleshoot site for help:

http://www.lifetechnologies.com/it/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting.html

Good "real" time!

Completely agree. Dilute your cDNA, you want it to come up 18-20 cycles otherwise you risk inhibiting your PCR because there is too much DNA. 

Hope it works!

Thank you all for your advices. I use ABI Step One machine. I have tried to readjust manually the backround signal and there is a slight improvement in some samples. As far as the dilution is concerned, I get the same sigmoidal curve enen though I use 1 ng/μl or 0.01 ng/μl DNA per sample (20-25 cycles). 

Agree with all of above

Not quite clear if you are doing DNA  PCR  (eg for quantifying fungal species) or RT-qPCR (expression)

1) try doing a serial dilution series (10x) followed by a melt curve. 18s is an abundant species. A high threshold is usually due to overloading. You may need to go down more than 1 order of magnitude to get good consistent amplification.

2) check products on gel - are they as expected

Hi Nikoleta I do not have experience with the kit you used (KAPA SYBR FAST ABI Prism qPCR Kit), or in general with the fast chemistry and I am sure that you follow correctly the manufactures instructions.

In the link that I send you in the previuos answer Is is said that mix well the components and centrifuge the 96-wells (or tubes), prior to cycling, is a critical step. So thaw well all the components, pay attention to the volume of diluition (do not pipette 1ul of DNA), mix very well the master mix and your samples, centrifuge.

As Ian suggest you can run the products on gel so you can check the PCR results

Don't give up and good luck!

Hi Nikoleta,

i suspect there are problems in the kit components. The first reaction being fine, the conditions should not be the suspect. Though i might have gone with Dr. Howe, regarding the background correction settings, the plot is way too beyond such explanation (the deltaRn of a sample in second experiment is too much). A melt curve analyis or simple loading on agarose gel could indicate whether the desired products were formed or not. If the desired products were not formed then you may change the kit components.  dilution should not be an issue because it worked in the first experiment, though minimum 1:10 or more should be good. 

18s RNA being in abundance, should be expected in early ct. so i guess the first one is correct (hence manual adjustments of background is necessary). Is this problem persistent, did you try other genes and get similar results. If that is the case either the machine or the kit components are bad.

Thank you for your help. Just to clarify, my work is to identify and quantify (if possible) four different species from proccessed samples. I have extracted DNA using Qiagen kit. My target gene is cytochrome b and I use 18s RNA as reference gene for building standard curve. I always test all my real time PCR in agarose gel and I get the desired product (one clear band without not specific bands). I get the sigmoidal curve even though I use cyt b or 18s RNA. I cannot understand why only once I managed to get a perfect amplification and after that  I couldn't repeat the same results. I get sigmoidal curve again and again.

Furthermore, when I tested the sample with the strange flat signal at the top, in agarose gel I got the desired product (one clear band without not specific bands).

Hi Nikoleta sorry to hear that you don't resolv your troubles.

I would ask you if you amplified, in the same plate, your DNA samples at different concentration, all your curves, from the less to the high concentrations, were sigmoidal? Do you try to amplify different DNA samples? Do you have another kit, also not fast but standard one, can you use other Syber kit with same samples and primers? Sorry for all this questions :) 

I attached an interesting  troubleshooting link that I find really useful for my experimets

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