Dear All,
I am currently trying to establish a methodology for measuring the amounts of a target gene (at the gDNA level) in my samples using qPCR (on a ABI 7900 machine using TaqMan reagents and primer/FAM-probe chemistry). However I have problems in that the levels of my targhet that I want to differentiate between are very close via qPCR - only across a couple of Ct's.
Therefore does anyone know of an established or reproducible way I can lower the efficiency of the qPCR reaction so that the differencies between samples will be greater? Does the addition of EDTA or alteration in salt concentration within the reaction change the reaction in a reproducible manner? If I do change these will this then invalidate the quantitative aspect of the qPCR?
Any help or advice would be much appreciated.
Many thanks,
Dominic
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3 replies to this topic
#2
Trof
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Posted 04 February 2011 - 02:59 AM
Honestly, I don't get it.
Are you measuring a copy number variation (amount of your target gene DNA in sample)?
In that case you just set the reaction, get the Cts and calculate the results. There may be small or big difference, but that's what you trying to find out. You can't say that the're too close, that just how they are (if you have optimised reaction without primer-dimers and so). You should of course calculate the results with efficiency correction.
High efficiency is a requirement for a quality results, in lower efficiencies the reaction just doesn't work well and who knows what happens there, it's definitely not suitable for quantification.
Or your problems is simply, that (both) the Cts are too high? That you can fix by using less primers.
Are you measuring a copy number variation (amount of your target gene DNA in sample)?
In that case you just set the reaction, get the Cts and calculate the results. There may be small or big difference, but that's what you trying to find out. You can't say that the're too close, that just how they are (if you have optimised reaction without primer-dimers and so). You should of course calculate the results with efficiency correction.
High efficiency is a requirement for a quality results, in lower efficiencies the reaction just doesn't work well and who knows what happens there, it's definitely not suitable for quantification.
Or your problems is simply, that (both) the Cts are too high? That you can fix by using less primers.
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#3
Maddie
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Posted 07 February 2011 - 11:15 PM
What do you mean by saying you want to differentiate your target?
I'm sorry, I don't get it either.
I'm sorry, I don't get it either.
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#4
drothwell
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Posted 08 February 2011 - 05:51 AM
Hi again,
Thanks for replying to the post. I don't want to 'differentiate' between results?
I have an assay to detect the level of a target gene in a sample which I can determine via a standard curve ranging from 100% positive down to 0%. However, most of my interest is looking at the 70-90% range of the standard curve, but here the differencies between samples are less than 1 Ct (the difference between 100% and 50% is 1 Ct). Therefore I was wondering if I could somehow alter the reaction to increase the difference between these higher concentration samples.
Having thought about it some more I really don't think it will be possible. Like you say the Ct is what it is and if I try and change the efficiency of the reactions it will compromise the quantitative nature of the assay.
Dominic
Thanks for replying to the post. I don't want to 'differentiate' between results?
I have an assay to detect the level of a target gene in a sample which I can determine via a standard curve ranging from 100% positive down to 0%. However, most of my interest is looking at the 70-90% range of the standard curve, but here the differencies between samples are less than 1 Ct (the difference between 100% and 50% is 1 Ct). Therefore I was wondering if I could somehow alter the reaction to increase the difference between these higher concentration samples.
Having thought about it some more I really don't think it will be possible. Like you say the Ct is what it is and if I try and change the efficiency of the reactions it will compromise the quantitative nature of the assay.
Dominic
