I have been having troubles with qPCR which frequently give signals in the mock samples. My experimental set up consists of samples that were infected with virus and mock samples which were not infected. When I run qPCR to see a viral gene expression, I sometimes get c(t) values between 30-35.Interestingly, the signal comes in one of those triplicates. I dont detect any signal in the negative control.I use AB sybergreen power/fast master mix.The melting curves always show up as one peak.The melting curves in the mock which are with c(t) 30 are above the baseline.But the ones with 35 are under the base line.When I run the same mock samples with two different primer sets that bind to viral gene, I sometimes get a low c(t) value for one and don`t get any signal or get at very late cycle for the other primer set.What could be the problem?
A ct between 30 and 35 suggest the target gene is not expressed, and is detecting only background, which is what you would expect from your mock fransfected cells. The fact that you detect the signal only in some of the replicates could reflect some detection variation. Make sure to run a background check before you run your experiment.
It could be but in that case i would see signal in the negative control and i also run my PCR products on agorose gel to see if it is an unspecific binding or dimerization.i couldn´t detect any. And also the melting curves appeared as one peak. ( Thank you) ;)
I whould consider the contamination ot the PCR products, and the nonspecifically amplified products.
What is the difference between your mock samples and negative control? If the negative control does not have cells, it could be some normal transcript in the mock sample amplifies under the right conditions, or they are infected with a related virus. Some people we work with have one set of diagnostic primers to check if virus is present and at what level, and then they run several sets of ID primers to make sure it is the correct virus.
A ct between 30 and 35 suggest the target gene is not expressed, and is detecting only background, which is what you would expect from your mock fransfected cells. The fact that you detect the signal only in some of the replicates could reflect some detection variation. Make sure to run a background check before you run your experiment.
All above suggestions especially Lambros and Paul solved your problem, Ct above 30 and 35 are considered as genes not expressed. When you run agrose gels, if you don't see any specific amplicon, it tells you there is no product made. You are okay, in my view.
You have to be careful with Ct some times 30 and 35 Cts doesn't mean the genes are not expressed, those genes might be expressed at low levels. It is good to take more cDNA for qPCR and amplify the product to see the change in this case.
hope you understood.
Best
RKG
when you run your qPCR do you also check in your samples for the expression of a house keeping gene such as Gapdh? If the delta Ct value between the house keeping gene expression and your virus genes is above 10 or 12 cycles, you can consider that the gene is not expressed. I suppose your negative control is a non template control (you add water instead of the cDNA) that is why you are not seeing any signal and it is an indication that your primers does not have dimers.
You see sometimes a signal in your mock samples because the genes seems to be expressed at very low levels and the machine at low expression levels gives you high variations even between triplicates.
Hope my answer was useful,
Good luck..
Why don't you increase the concentration of your cDNA templates to get lower CT values, out of the background area (~35CT). For the amplification in the mock, whether you already check potential primer cross/homodimers, you may have to increase the annealing and/or reading temperature of your cycling conditions.
Hope you'll fix your problem.
All the best.
Thank you very very much...All comments are really informative. I attached a document includes my ct values. In order to test my mock cells, I used to same mock cells which were from two different experiments but they are exactly same cells, from same stock and treated in a same way.However, the results are not consistent. I should point out that I used two different sybergreen; one is power one is fast. I am always using the PCR programme which the company suggests. This is another concern I actually have. Because all my pcr programmes have 60°C as a melting temperature eventhough I had different primers with different melting temperatures on the same assay. However, all melting temperatures appears as one peak. I run the products on agorose gel and saw only one band at correct size; so no primer dimers or unspecific product and of course I didnt observe bands in mock. Another strange thing, when I run in triplicates, as i dont see any signal in two of them, I can see in one of them. Is that pipeting error or else? I never actually change the machine adjustments, baselines,threshold so on they all automatically were adjusted. Do you suggest me for example manually adjust the base line? I am looking forward to seeing more comments and suggestions. Thank you very much once more ;)
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