Does error (difference of less than 3 degree Celsius) in annealing temperature have any role in getting a higher Cq/Ct?
Charles,
The short answer is that it is by no means necessary to exclude Ct values over 30. This can be due to a variety of reasons, including that your target is expressed at relatively low levels, the efficiency of your assay (TaqMan or Sybr primers) is low owing to poor design or reaction conditions, you are experiencing inhibition of the reaction due to some contaminant etc.
Even Ct values that are higher (35 and above) are not "compulsory" to omit from your analyses, but you'll notice that there will be less agreement between technical replicates in these cases. This is usually because you're pipetting a small number of target molecules amongst technical replicates, and the statistical sampling error associated with that pipetting event becomes increased (i.e. the Poisson error).
Well, I would not compulsorily exclude them. There are two possibilities why you get very high Ct values:
If you are working with your own primers, you might have not found yet the best cycling conditions (two-step vs. three-step cycles, annealing temp). This you fix by testing the conditions on 5x or 10x serial dilutions (minimum 4 dilutions) and checking the linearity of the results and the efficiency of the PCR (if Eff
Thanks a lot for your answer. I doubt about the annealing temperatures of the genes. Will a difference of 2 to 3 degree Celsius make a huge difference in Ct value? For Beta-actin the Ct value is around 23. So I guess I have enough cDNA.
Best way to know if you should consider value over 30 is by running appropriate controls.
1) Run real time PCR with RNA of the sample giving you value over 30. If you get amplification around 30, you can assume that it is background amplification.
2) Check if two fold dilution of sample results in the Ct value going up or down by 1.
Charles,
The short answer is that it is by no means necessary to exclude Ct values over 30. This can be due to a variety of reasons, including that your target is expressed at relatively low levels, the efficiency of your assay (TaqMan or Sybr primers) is low owing to poor design or reaction conditions, you are experiencing inhibition of the reaction due to some contaminant etc.
Even Ct values that are higher (35 and above) are not "compulsory" to omit from your analyses, but you'll notice that there will be less agreement between technical replicates in these cases. This is usually because you're pipetting a small number of target molecules amongst technical replicates, and the statistical sampling error associated with that pipetting event becomes increased (i.e. the Poisson error).
Hi Charles, I see no one really answered your question about the 2-3 °C in PCR... not sure what you really mean, anyway, it is difficult to say yes or no in general, but if a PCR is not optimal, a 2-3 °C shift can change things radically.
But you should run your product on gel and see how it looks: if it's clean but weak, you can decrease 2-3 °C the annealing and you might increase your yield... but you might also increase unspecific! If your amplification is already smeared, or with (multiple) unspecific bands, then you know why your specific band is weak. In this case raising the temperature might help, elongating the annealing time might help (thermodinamically a specific binding is normally more stable than an unspecific, but you also have to consider the kinetics of the binding, some primers bind slowly, while unspecific binding is almost always a quick on-off, therefore giving more time for the annealing builds up specific annealing vs. unspecific), but you might also need to simply change primer pair.
By the way, actin mRNA is probably the most abundant mRNA in the cell. If you are not diluting your cDNA at least 10 times and using 0.5 ug of RNA for your RT the Ct is not particularly good... So, it would be ok if your gene would come out also in a similar range, but in your specific conditions it might want some improvements on the RNA and/or RT side...
Good luck
@Pietro Pilo Boyl
The problem I am facing is little different. I did gradient PCR with cDNA for a set of primers to find out its annealing temperature. For that particular set of primers the calculated Ta (Tm-5 degree) was 45 but in gradient PCR it didn't get amplified in that temperature. It didn't even work temperatures near to that. So I decided to keep it in a Real time PCR reaction with annealing temperature 49 along with other genes. In this it gave Cq/Ct value of around 32. So now I am confused whether to take this value for further analysis.
I see Charles... let me suggest you something... change the primers. Such a low annealing temperature is very dangerous, you want to be at least over 55 °C, better around 58-60 °C, which means 50-60% GC content in the primer. Moreover, I always suggest when designing primers to have at least 3 G,C bases in the last 5 nucleotides at the 3' end of your primer, possibly ending your primer with GG or CC: this is where the Taq polymerase starts elongating and the free energy (dG ) of the 3' end of the primer (last 7-8 bases) should be around -13 to -15.
Anyway, for your reassurance, run your qPCR reaction at 49 °C on the gel... and check what you have there... I don't think you'll see a single band of the expected size of the amplicon...
Hi Charles,
I am not too much of an hands on expert, but we had a similar question in out lab and you could put your amplification product on a gel. If you can find a band you have product if you get a smear it is more likely to be background.
Similarly you can run a dilution experiment as suggested already. You take sample that works well and dilute it down that should give you some indication about your lab settings, sample handling, quality of enzymes .......
KR
Jens
http://www.biomarker-discovery.com/
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