I've determined the LOD/LOQ value for a qPCR assay (1 primer pair, 1 taqman probe) and it's higher than expected. I've been trying to figure out why - i've tried to find information about this but all I can find online is explanations of what the LOD is or reporting LOD values from different assays.
Could anyone point me in the right direction?
Thanks
Hi,
could you add some more details what is the value and what did you expect and why? This is pers e hard to say, could be as easy as your quant standards being not appropriate.
Sven
So it's a new assay that was created so all of these things are being done for the first time, no standards have been used. The lod is around 3000 copies per reaction (determined using g block synthetic DNA) and similar assays have a much better value of around 10.
Thanks
hello ..
The limit of detection (LOD) and limit of quantitation (LOQ) of a qPCR assay refer to the lowest concentration of target DNA that can be reliably detected or quantified, respectively, using the assay. There are several factors that can affect the LOD and LOQ of a qPCR assay, including the sensitivity of the qPCR instrument, the efficiency of the qPCR reaction, the quality and quantity of the DNA template, and the design of the primers and probe.
If your LOD/LOQ values are higher than expected, there are several potential reasons why this might be the case:
Insufficient sensitivity of the qPCR instrument: If your qPCR instrument has a low sensitivity, it may be unable to detect or quantify low levels of target DNA.
Poor qPCR reaction efficiency: If the efficiency of the qPCR reaction is low, it may be difficult to detect or quantify low levels of target DNA. Factors that can affect qPCR efficiency include the quality and concentration of the primers and probe, the purity and concentration of the DNA template, and the presence of contaminants or inhibitors in the reaction.
Poor quality or quantity of the DNA template: If the quality or quantity of the DNA template is poor, it may be difficult to amplify low levels of target DNA.
Poor primer and probe design: If the primers and probe are not well-designed, they may not bind efficiently to the target DNA, resulting in low amplification and detection of the target.
To troubleshoot the issue with your qPCR assay, you may want to try optimizing the reaction conditions (e.g., adjusting the concentrations of the primers and probe, optimizing the DNA template concentration) or designing new primers and probe. You may also want to consider using a more sensitive qPCR instrument if possible.
My regards
Hey Elise. If your work is Novel( everything is new),Then you need to ensure that everything is ok. Start with your extraction method( 260/280 ration and concentration). try to use Gel-based PCR and observe the bands with different dilutions of your template. Then jump to QPCR and apply 10-folded serial dilutions as standards ( alone in the reaction) and pay attention to P , E and squared R values of the reaction. N.B: use snapgene or other softwares to check ur primes and probes and pay attention to Tm for all.
There are several factors that can influence the limit of detection (LOD) and limit of quantification (LOQ) values of a qPCR assay. Some potential causes of high LOD/LOQ values in a qPCR assay include:
Thank you all for your responses!
As a follow up, how do you know if the assay is badly designed? and if there is low enzyme efficiency?
You can use your Current MasterMix for any other Well-Designed reaction. So the new reaction will have your MasterMix and of course your pol enzyme( but not ur Primers and probe). Then you need to observe the E values (above 0.85 is good) and R square value ( above 0.99980) is perfect. of course you need around 5 10-folded serial dilutions to measure these values with good precision. Remember if your mastermix (and enzyme) is good you need to check primers and probe for your original reaction.
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