Improving qRT-PCR detection limits - (May/12/2011 )
Hi guys,
I am trying to design a real-time PCR assay for copy number determination of an antibody gene in mammalian cells, using TaqMan fluorescent probes.
The issue is, I need to find a way to improve the sensitivity of the assay. So far, it works perfectly in the range 10^8 – 10^4 copies of either antibody chain per reaction (acceptable efficiency, Pearson correlation, amplification is every three cycles). When 10-10^3 standards are added to the standard curve, however, they all cross the threshold line at about 24 cycles along with the 10^4 standard, irrespectively of the fact there is far less DNA in them, and they should cross the line a lot later.
So, far, we have tried optimising primer concentrations and cycling conditions, tried different primers, ran the assay on a different machine…. The problem still exists.
Any bright ideas or suggestions on what to try next?
Thanks,
Hriss
Hriss on Thu May 12 10:23:31 2011 said:
Hi guys,
I am trying to design a real-time PCR assay for copy number determination of an antibody gene in mammalian cells, using TaqMan fluorescent probes.
The issue is, I need to find a way to improve the sensitivity of the assay. So far, it works perfectly in the range 10^8 – 10^4 copies of either antibody chain per reaction (acceptable efficiency, Pearson correlation, amplification is every three cycles). When 10-10^3 standards are added to the standard curve, however, they all cross the threshold line at about 24 cycles along with the 10^4 standard, irrespectively of the fact there is far less DNA in them, and they should cross the line a lot later.
So, far, we have tried optimising primer concentrations and cycling conditions, tried different primers, ran the assay on a different machine…. The problem still exists.
Any bright ideas or suggestions on what to try next?
Thanks,
Hriss
sounds like you have contamination of your standards with target template. this can happen if you handle your template in very high concentrations (like your 10^8 copies standard). What is your standard? Plasmid sequence? Are you using filter tips for preparing your standards (and i hope you are preparing your standard curves not on your PCR pipetting place with the same pipetts)? how is your non template control looking?
sounds like you have contamination of your standards with target template. this can happen if you handle your template in very high concentrations (like your 10^8 copies standard). What is your standard? Plasmid sequence? Are you using filter tips for preparing your standards (and i hope you are preparing your standard curves not on your PCR pipetting place with the same pipetts)? how is your non template control looking?
Thanks for the quick reply.
My standard is a plasmid sequence, yes. I am not entirely sure whether it is a contamination with template, because negative and no-template controls are negative, so the issue is not the reaction mixture. I am setting up the mastermixes in a VLF in a different room to the one with the template, I am UV-ing pippettes etc.
I have tried constructing a standard curve on the bottom end of the dynamic range only: 10-10^4, to eliminate the possible influence of any high template concentration standards, but unfortunately the result was the same.
Could it be that I have some kind of inhibiting agent that I am aware of, or could I be working under suboptimal conditions that allow detection of high copy number but not low copy number?
Thanks,
Hriss