Hello colleagues,
I have designed some TaqMan probes for using in ddPCR, and to save expensive ddPCR reagents I decided to test the probes in conventional RT-PCR machine first. The probes are labeled with FAM and JOE, and the quenchers are BHQ-1 and BHQ-2, respectively.
Our RT-PCR machine is AB7500, which is quite old. It doesn't have BHQ options for quencher in its software. I only can choose between TAMRA, MGB and "no quencher". Which of these options is more appropriate if I actually have BHQ? Is this parameter important at all, since I calibrated the PCR machine using fluorescent dyes, but no quenchers were needed for calibration? Is it possible to reprocess raw fluorescence data with correction for another type of quencher after the reaction is finished?
P.S. BHQ quenchers are definitely suitable for our ddPCR machine and its software, so there would be no such problem.
Dr. Chesnokova,
I've worked with TaqMan probes with dark quenchers for some time now. While I'm not familiar with your specific instrument, I do work with an Applied Biosystems StepOnePlus (same manufacturer). The reason I bring this up is becuase the quencher list you mention is similar to the one I see: "NFQ-MGB," "None," and "TAMRA."
In my experience, I have never encountered a TaqMan probe that uses a non-fluorescent quencher (NFQ) but not a minor groove binder (MGB) modification and vice-versa. This leads me to believe that many may use these terms interchangeably, and that if you choose "MGB," your instrument will actually interpret "NFQ-MGB." It doesn't make sense that MGB would be an option on its own, given that this modification provides no quenching potential on its own.
On another note, I think that if you choose "No Quencher," your instrument may assume you're running a SYBR green reaction, and the moment at which it reads will be inaccurate. And selecting TAMRA would simply yield unreliable readouts.
Thank you Jose,
but I have BHQ (black hole quencher), not NFQ, so it's not a synonim to MGB.
I also guess that in any case the instrument will not confuse probes with SYBR green, since I specify the fluorescent dyes during setup, and each dye has its own calibration. However, I agree that improper reaction setting may cause unreliable readouts.
I already performed the reaction using "MGB" option, found out that I can change this option afterwards and nothing changes (while if I change the fluorescent dye or standards concentration afterwards, results change dramatically), and established that two of my probes are working and other two are not.
As far as I know, "Non-fluorescent quencher (NFQ)" or "dark quencher" is the term used for all quenchers that don't emit a signal of their own. A black hole quencher (BHQ) is only one type of NFQ, so it's covered under this category. Others include Dabsyl, Qxl quenchers, Iowa black FQ, Iowa black RQ, IRDye QC-1.
http://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2015/04/07/qpcr-probes-selecting-the-best-reporter-dye-and-quencher
I would continue using the MGB selection, and perhaps try and contact Applied Biosystems for confirmation that this is the right choice.
I'm not so sure, so I wouldn't argue with you about the difference between BHQ and NFQ.
But I must say that BHQ can be used without MGB - please see the graph in the bottom of this page: https://www.thermofisher.com/ru/ru/home/products-and-services/product-types/primers-oligos-nucleotides/applied-biosystems-custom-primers-probes/custom-taqman-probes/taqman-mgb-probes.html
It says "non-MGB assay (FAM dye–labeled, with BHQ)".
Our machine is a AB 7500 too. I've been using probes designed with BHQ-1 and they work perfectly when NFQ-MGB is selected
- Badges
- Science method