The same primer pairs with cDNAs, reverse-transcribed from different plant tissues, give different amplification rates which are almost always above 2. Can these data be used or I should optimize the PCR conditions?
From the plot you show your the reason for your problem is obvious!
The ct values (in the y-axis) are supposed to decrease linearily with the log concentration (on the x-axis).It is more convenient to look "from right to left", so with increasing dilutions factors the ct values should linarily increase.
They do so, approximately, for -2 < x < 1. However, they do not continue to increase with the same rate for lower concentrations (-4 < x < -2). This is extremely obvious when looking at x=-3 and x=-4, where the ct values do not increase at all. Au contraire, most of the ct values at the highest dilution (x=-4) are even smaller than the ct values of the next lower dilution (x=-3). This is likely explained by the amplification of unspecific products and primer-dimers.
Your mistake is that you did not recognize the limitation of the "linear range" of your assay (somewhere between x=-2 to x=-3). You used all the points to fit the straight line. This line will have a small slope because the ct values at x=-2 and x=-3 are "too low". The smaller the slope the higher is the apparent efficiency.
You youd use only the values in the range -2 < x < 1 to fit the line and to determine the efficiency. I recommend that you prepare more dilution steps (you used 6 different 1:10 dilutions, I would suggest using 17 1:2 dilutions instead. This would cover the same range of concentrations but give you a far better resolution, a better information about the limit of quantification, and a more precise estimate of the efficiency).
Are you using a SYBR based assay? An amplification rate higher than 2 would probably indicate that you have primer dimer products and/or non specific priming. You should run your products out on a gel to see if there are multiple bands and do a dissociation curve analysis. Non-target amplification could come from other sites in the cDNA (in which case you'd want to optimize your primer sequences, primer concentration, or Tm) or it could be coming from contaminating template - do you have no-template control wells? If so, do they show any amplification?
It would help if you show the data used to determine the efficiency. Also, showing some representative amplification curves would be helpful.
First of all, thank you for helping, because I perform qPCR experiments for the first time, so I don't know all pitfalls.
Govinda Sharma, yes, I am using an intercalating dye-based assay and there is no amplification in no-template control wells. Apparently, it is linked with non specific amplification, I will run the products out on a gel.
Jochen Wilhelm, the data used to determine the efficiency in attaching. Efficiencies of different praimer pairs (denoted in yellow boxes) marked with magenta.
Do I understand rightly that if where is no non-specific amplification, the amplification rate for particular primer pair should always below 2 and should be the same every time despite origin of templates?
From the plot you show your the reason for your problem is obvious!
The ct values (in the y-axis) are supposed to decrease linearily with the log concentration (on the x-axis).It is more convenient to look "from right to left", so with increasing dilutions factors the ct values should linarily increase.
They do so, approximately, for -2 < x < 1. However, they do not continue to increase with the same rate for lower concentrations (-4 < x < -2). This is extremely obvious when looking at x=-3 and x=-4, where the ct values do not increase at all. Au contraire, most of the ct values at the highest dilution (x=-4) are even smaller than the ct values of the next lower dilution (x=-3). This is likely explained by the amplification of unspecific products and primer-dimers.
Your mistake is that you did not recognize the limitation of the "linear range" of your assay (somewhere between x=-2 to x=-3). You used all the points to fit the straight line. This line will have a small slope because the ct values at x=-2 and x=-3 are "too low". The smaller the slope the higher is the apparent efficiency.
You youd use only the values in the range -2 < x < 1 to fit the line and to determine the efficiency. I recommend that you prepare more dilution steps (you used 6 different 1:10 dilutions, I would suggest using 17 1:2 dilutions instead. This would cover the same range of concentrations but give you a far better resolution, a better information about the limit of quantification, and a more precise estimate of the efficiency).
Jochen Wilhelm, thank you very much! I've never thought this way, today I will definitely retry the estimation of the efficiency using 1:2 dilutions. If it is fine, will the primers be proper for further experiments with templates from other tissues without reestimation for every template sourse?
- Badges
- Science method