qPCR High variation well to well

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cubita1
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qPCR High variation well to well

I have big problems because i am doing a standard curve and  I am facing a high variation well to well. I would appreciate any sugestion..
 

Ivan Delgado
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Hi cubita,
High variation from well to well can be a result of many things. Some you may want to consider: 
1. Is your block dirty? You should be able to check any block contamination using the instrument's software
2. Is your pipetting technique adequate? Remember that qPCR master mixes are viscous so it is advisable to do reverse pipetting
If you provide more details about your situation we may be able to point out other sources of possible problems

cubita1
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Dear Ivan

Dear Ivan
Thanks for your advices. I will try the reverse pipetting.
Another problem that I have is that the well with the largest amount of cDNA does not have the biggest signal of fluorescence.
 

Ivan Delgado
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Hi cubita,
Real time PCR is very sensitive to inhibitors. If you see a lower signal in your highest cDNA sample, then it is possible that you have too much cDNA. Typically any signal that comes up earlier than cycle 10 - 15 is too high. One way to get around this is to manually set your baseline to a lower range of cycles, at least 2 cycles below the cycle at which your first signal comes up in your experiment.  

cubita1
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Dear Ivan,

Dear Ivan,
Thanks for your advices. I prepared a standard curve with a plasmid, using different amounts of DNA. I obtained nice curves from cycle around 14 to 30, with the required space between them and a very low variation from well to well. After the cycle 30, I have another reality: The space between the curves is not the expected and also there is a high variation from well to well. I have prepared the dilutions many times, and the problem is the same, even using vortex to mix well the solutions. In addition, I use to prepare a mix for no template control and I divide this mix in 3 wells. I am surprised because I have obtained different signals for the 3 controls and one of them with a fluorescence signal above the threshold. I am really confused. Why my curves are Ok only in a range of cycles?
 
    
 

Ivan Delgado
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Hi Cubita,
What you got is not uncommon. Typically Ct values higher than 35 are questionable since you are starting to amplify noise. The high variation is possibly due to the Monte Carlo effect, which in simple terms states that when you have very little template sometimes it gets amplified and sometimes it does not. In other words, your high variation is due to very low template concentration in your qPCR reactions that leads to higher variation in signal. Unfortunately there is little you can do about this other than design a new assay (or spend some quality time pushing to optimize the assay even further). 
I am not sure what you mean by "fluorescence signal above the threshold". Your negative controls should give you close to no signal. If you are getting signal it could be due to contamination or other issues. 

cubita1
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Thanks Ivan,
I can see that I am having 2 problems. First the very low template concentration in the PCR and second, a low contamination :(
I know how to fix the problem of contamination. However, now I don’t know how to detect low expression in my samples accurately.
 

Ivan Delgado
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Hi cubita,
Detecting low concentrations of DNA by qPCR has a lot to do with your assay design. If your assay is detecting a single gene in your DNA, then you are limited to detecting a single site per DNA molecule. Yet, if you are trying to detect a repeat, then you can increase the sensitivity of your assay significantly. For example it is typical to design assays to detect the 16S region since this genes are found as multi-copy genes in bacteria. Likewise in humans you can design assays to detect repeat regions (like Alu regions), which can have over 100,000 copies in a single genome. It depends on your experimental design and what you are trying to detect.

Nikail Collins
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ROX

Does your mastermix include a passive dye such as ROX? It can help with well to well variability.