Efficiency greater than 100% using taqman probes? - (Nov/15/2006 )
Hi there,
I wonder if anyone else has experienced this?
I understand very well how an E can be greater than 100% with a sybr green method - primer dimer, unspecific annealing, etc.
But how can this happen with a taqman assay?
My E is 2.29 (and the values look perfect, the r2 value is 0.998).
I am really confused! Any ideas?
I wonder if anyone else has experienced this?
I understand very well how an E can be greater than 100% with a sybr green method - primer dimer, unspecific annealing, etc.
But how can this happen with a taqman assay?
My E is 2.29 (and the values look perfect, the r2 value is 0.998).
I am really confused! Any ideas?
Smurray,
Why are you thinking the primer dimer problem, as well as the unspecific priming occur in SYBR green reactions only? In real-time, SYBR green or TaqMan are only ways of detecting the amplicons. Of course there are differences in that SYBR green binds to ANY dsDNA, while hydrolysis probes bind specifically to the target (between the flanking sequences where your primers align).
Primer dimers can form based on their intrinsic structure that you have to check before choosing a design or another. The efficiency will modify in the presence of those secondary products.
When you're getting efficiency numbers greater than 100%, that usually indicates a saturation of the RT or the PCR, and you see less of a delta Ct between higher dilutions. That can sometimes throw off the slope on that high end and create artificially high efficiency. It is very important when you are looking at your standard curves to look for even spacing between all of them. If you do see compression on the upper end, throw out those points and recalculate the efficiency. Or do it again, more carefully.
Despite the fact that electrophoresis appears "old fashion" technique as compared to real-time PCR, IT IS recommended to check your amplification products by gel electrophoresis and see results. This replaces NOT the melting curve analysis, it's complementary to it.
Hi there,
The reason I thought it couldn't be primer dimer is because of the probe - the probe wouldn't bind to primer dimer, presumably, so wouldn't give off fluorescence signal. Am I wrong in thinking that?
About the saturation of the PCR, that is possible, as I made the dilution series using PCR product and my Ct values were all quite high. I can certainly dilute more and try again.
thanks
[/quote]
Smurray,
Why are you thinking the primer dimer problem, as well as the unspecific priming occur in SYBR green reactions only? In real-time, SYBR green or TaqMan are only ways of detecting the amplicons. Of course there are differences in that SYBR green binds to ANY dsDNA, while hydrolysis probes bind specifically to the target (between the flanking sequences where your primers align).
Primer dimers can form based on their intrinsic structure that you have to check before choosing a design or another. The efficiency will modify in the presence of those secondary products.
When you're getting efficiency numbers greater than 100%, that usually indicates a saturation of the RT or the PCR, and you see less of a delta Ct between higher dilutions. That can sometimes throw off the slope on that high end and create artificially high efficiency. It is very important when you are looking at your standard curves to look for even spacing between all of them. If you do see compression on the upper end, throw out those points and recalculate the efficiency. Or do it again, more carefully.
Despite the fact that electrophoresis appears "old fashion" technique as compared to real-time PCR, IT IS recommended to check your amplification products by gel electrophoresis and see results. This replaces NOT the melting curve analysis, it's complementary to it.
[/quote]
[quote name='smurray' date='Nov 17 2006, 08:21 AM' post='77396']
Hi there,
The reason I thought it couldn't be primer dimer is because of the probe - the probe wouldn't bind to primer dimer, presumably, so wouldn't give off fluorescence signal. Am I wrong in thinking that?
About the saturation of the PCR, that is possible, as I made the dilution series using PCR product and my Ct values were all quite high. I can certainly dilute more and try again.
thanks
[/quote]
Smurray,
Why are you thinking the primer dimer problem, as well as the unspecific priming occur in SYBR green reactions only? In real-time, SYBR green or TaqMan are only ways of detecting the amplicons. Of course there are differences in that SYBR green binds to ANY dsDNA, while hydrolysis probes bind specifically to the target (between the flanking sequences where your primers align).
Primer dimers can form based on their intrinsic structure that you have to check before choosing a design or another. The efficiency will modify in the presence of those secondary products.
When you're getting efficiency numbers greater than 100%, that usually indicates a saturation of the RT or the PCR, and you see less of a delta Ct between higher dilutions. That can sometimes throw off the slope on that high end and create artificially high efficiency. It is very important when you are looking at your standard curves to look for even spacing between all of them. If you do see compression on the upper end, throw out those points and recalculate the efficiency. Or do it again, more carefully.
Despite the fact that electrophoresis appears "old fashion" technique as compared to real-time PCR, IT IS recommended to check your amplification products by gel electrophoresis and see results. This replaces NOT the melting curve analysis, it's complementary to it.
[/quote]
[/quote]
Probe hibrydize with the target only, indeed, but primer-dimers, although not detected, still affect the efficiency numbers. Also the additional products (I've got that when working with FAM becons probes and the target was too much concentrated: gel electrophoresis always showed the main expected band together with several 3-5 additional weaker bands above the desired one; these disappeared with a more diluted template)
Hi there,
thanks for the reply.
Ok so primer dimer can affect the efficiency... I would imagine, if primer dimers were forming a lot, shouldn't the efficiency of the target reaction decrease, not increase?
I'll run my samples on a gel and have a look to check.