I am getting large SD between the CT of the target gene technical replica. Can anyone explain this? The SD of the control Ct value is very small as expected.
Tell us more about the experiment please.
Short answer is that technical replicates should be the same - that's why you do em, right!. Test your plate layout (randomise sample and replicates), add more controls to the experiment. Increase the number of technical replicates. Ensure your pipe tying is tip top!
Hi,
the SD of Ct values depends on the Ct itself. Higher Ct values show larger SD. Thus if your control has a lower Ct than your target this may be the reason.
Hi Abeer
Ensure you pipe the samples very well, in my opinion in most of the time bad pipetting is the main reason for this variation
good luck
Let me give you more details. I make master mix for the target samples with the target primer and also do the same for the Endo gene (control). I make three replicate from each plant. Then from the master mix I divide what's enough for three replicas and then add the cDNA.
If it is pipetting error it will occur within the Endo technical replica sometimes, but this doesnot happen. It noticed only within the target replica. Could 've been the primer. Should I design new one?
Norman above mentioned that if your Ct values are high (above 30), you will naturally see larger SD between technical replicates (it is the nature of the stochastic beast that qPCR is; the higher the Cq value is above 30 or so, the more Poisson noise is associated with your signal). However, a better way to make a technical triplicate is to add your cDNA to the mastermix tube before you split it into the 3 final wells. This way, you have a true technical triplicate replicate without introducing more undo error into the process of setting up the reactions piece-meal in the plates as well.
What are some typical values for your target replicates? E.g. [35.1, 37.8, 36.4]? If so - that would be expected among Cq values in that range, etc.
If you are getting target replicates of [24, 25.3, 26.1], then there is a problem.
Don't forget about primer dimer (and other non-specific) contributions to signal... are your melt curves clean? Meaning, of all the target reactions you run and get a signal for, is the dissociation peak specific for your target?
I agree with Jack, it is better to prepare master mix with your cDNA and then divide it into replicates' wells and see what will be happen.
i have also see such problem in case of gene with low expression level. how did you optimized the concentration of target gene for your PCR? did you perform serial dilution and analyzed the efficiency of your primers?
As jack asked you, would you please inform us about PCR condition in detail? ct of triplicate gene of interest and control?
hope to cope with this high SD with each other :)
With robust assays generating Cq values of ~30 and below, generally less than or equal to 0.2 Cq unit variation is acceptable among technical replicates. But, as Poisson noise wages greater influence the less and less target there is, this value can vary up to 1.5 Cq units or more. It has even been observed that 1 copy reactions (depending on efficiency of amplification) can appear anywhere from 35 (Vandesompele) to 48 cycles (Otto). So, it is the very presence of large variation in technical replicate Cq values (based on nil reaction analysis) that provides the signature of single-copy reactions (and is the basis of droplet digital PCR). So, you see, there is no "one answer applies to all Cq values" here. It depends on how much target template there is in the reaction, and then, from there, it depends on how and when Poisson noise begins to impact the entire situation...
i am also facing the same problem, my primer is the same for all samples, the only difference is cDNA. First i made the master mix (added every thing including cDNA and primers as well) and then distributed in the plate . one sample (means one kind of cDNA sample) have standard error of less than 0.01 but the other one have very high standard error. all the sample were prepared at the same time and distributed in the same qPCR plate. what is the possible reason for getting high standard error in second sample ?
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