In order to wait for a more complete article about inhibition control and strategies to have, commercial kits are available on the market.
Historically, to my knowledge, Applied Biosystem was the only one several years ago to sell a commercial IC (Inhibition Control) allowing to monitor inhibition in duplex qPCR.
Historically, to my knowledge, Applied Biosystem was the only one several years ago to sell a commercial IC (Inhibition Control) allowing to monitor inhibition in duplex qPCR.
Its little nickname?
TaqMan Exogenous Internal Positive Control.
This reference works well having tested it in my lab, with a VIC grafted probe not to interfere with FAM grafted fluorophore usually used on most target probes in TaqMan qPCR. However, the use of this kit implies to realize a color compensation between FAM and VIC grafted probes. Indeed, a spectra overlapping does exist and if not done, part of VIC signal is found in FAM signal and can lead to false results. It will be detailed in further posts.
Even if called "Internal", Applied IC is totally chimeric in reality and has no match with the target system. Primers used for IC amplification are not the same as for target one. It seems to be obvious but has to be noticed anyway. It is part of external non competitive IC.
This reference works well having tested it in my lab, with a VIC grafted probe not to interfere with FAM grafted fluorophore usually used on most target probes in TaqMan qPCR. However, the use of this kit implies to realize a color compensation between FAM and VIC grafted probes. Indeed, a spectra overlapping does exist and if not done, part of VIC signal is found in FAM signal and can lead to false results. It will be detailed in further posts.
Even if called "Internal", Applied IC is totally chimeric in reality and has no match with the target system. Primers used for IC amplification are not the same as for target one. It seems to be obvious but has to be noticed anyway. It is part of external non competitive IC.
It does react well to different kind of inhibition despite a pretty strong own target DNA concentration(Ct around 31-32), leading possibly to sensitivity problems on the target when in small quantities.
Two recent references has appeared :
- Eurogentec : Universal Exogenous qPCR Positive Control
- Qiagen : QuantiFast Pathogen +IC Kits
I did not test many times the Eurogentec kit, working not pretty bad. It uses Yakima Yellow fluorophores instead of VIC compared to previous one.
Since two years, I started to use to Quantifast pathogen+IC kits. This kit works really well and that is the one I still use now. Once you have made the color compensation (FAM-VIC), it is done for every PCR using this solution. Sensitivity to any inhibitors is good to my experience in environmental matrices.
Here was few informations about commercial IC in qPCR, I will try to do a longer post about inhibition monitoring strategies later.
Have good amplification....
Two recent references has appeared :
- Eurogentec : Universal Exogenous qPCR Positive Control
- Qiagen : QuantiFast Pathogen +IC Kits
I did not test many times the Eurogentec kit, working not pretty bad. It uses Yakima Yellow fluorophores instead of VIC compared to previous one.
Since two years, I started to use to Quantifast pathogen+IC kits. This kit works really well and that is the one I still use now. Once you have made the color compensation (FAM-VIC), it is done for every PCR using this solution. Sensitivity to any inhibitors is good to my experience in environmental matrices.
Here was few informations about commercial IC in qPCR, I will try to do a longer post about inhibition monitoring strategies later.
Have good amplification....
