Do you use the passive reference dye in DNA-binding-dye type real-time PCR?
Hi Jochen,
I have not compared myself assays with and without ROX.
As far as I know though, it largely depends on the instrument that you are using eg ABI instrument (at least the older ones like ABI 7500) required ROX passive dyes but on the other hand there are instrument that can function without ROX dye eg. BioRad, QIAGEN, Eppendorf, Roche, Agilent. A passive dye like ROX dye is believed to provide an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or volume. Since, if I understood correctly, you are planning to use SYBR green as a reporter dye and you wont be needing multiple wavelengths for multiplex probes there is no harm in trying it. If you have a kit for Real Time PCR it will either have ROX dye already into the mastermix or it will provide a separate vial for you to add it into your reaction. The kit also should provide the dilution of ROX that you should use according to your instrument.You check out for example the datasheet from the kit I am using. It is quite comprehensive in terms of instruments and ROX dye requirements. You can also have a look at the web site of Affymetrix for actual ROX concentrations in the final reaction.
http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131187#1_1
Best of luck with your assays!!
Iliana
http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131187#1_1
Iliana, could you give a little more information? What are the differences? Have you compared assays with and without ROX?
Hi Jochen,
I have not compared myself assays with and without ROX.
As far as I know though, it largely depends on the instrument that you are using eg ABI instrument (at least the older ones like ABI 7500) required ROX passive dyes but on the other hand there are instrument that can function without ROX dye eg. BioRad, QIAGEN, Eppendorf, Roche, Agilent. A passive dye like ROX dye is believed to provide an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or volume. Since, if I understood correctly, you are planning to use SYBR green as a reporter dye and you wont be needing multiple wavelengths for multiplex probes there is no harm in trying it. If you have a kit for Real Time PCR it will either have ROX dye already into the mastermix or it will provide a separate vial for you to add it into your reaction. The kit also should provide the dilution of ROX that you should use according to your instrument.You check out for example the datasheet from the kit I am using. It is quite comprehensive in terms of instruments and ROX dye requirements. You can also have a look at the web site of Affymetrix for actual ROX concentrations in the final reaction.
http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131187#1_1
Best of luck with your assays!!
Iliana
http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=131187#1_1
Thank you very much Iliana and Jochen for this helpful discussion. Jochen actually raised the question really bothering me - whether there is a true difference when using and not the reference dye. And the reason is simple - crossing russian border curiously causes the five-fold increase in the prices... Though, economizing on necessary things is far more expensive, thus I'll have to use fitc (bio-rad machine).
Iliana, thanks for the link and the instruction - I need to build up my own PCR mix, so the information on the content and the concentrations is very valuable.
Inclusion of ROX in RT-PCR can normalize the non-PCR related signals and provide a baseline for the fluorescence to begin.
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