Hello Leonid,

In a general sense, q(RT)-PCR is used as a way to quantitatively look at the expression as a function of mRNA  present relative to some other gene - typically using something that should be constant as a reference point. Meaning you want to see, based on some treatment, what effect that has on the level of expression in your system.

In practice there are various ways to conduct a qPCR experiment. A simple general workflow is the use of a fluorescent molecule and a primer specific for each gene you're interested in comparing. For instance, in the TaqMan assay endonuclease activity of the polymerase cleaves the gene specific primer that has a fluorophore and quencher attached.The cleavage releases the fluorophore from the quencher and allows emission by the fluorescent molecule producing RFUs as a function of mRNA present for that gene specific primer.

ROX is a fluorophore that can be used as a reference dye to normalize fluorescence across all of your samples (experimental and controls) for your instrument providing you a baseline. Essentially you just include a small, constant amount in all of your samples as a control.

Hope that helps.

OK, got it. It just normilises the background fluorescence. Thank you !

This is an excellent description of the role of a passive reference:

http://www.bio-rad.com/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye?ID=MW472W15

Thank you!

In simplest definition machines that need a passive flurophore normalization work on single optic fibre system on which florescence from each well is reflected on. Rox helps to minimize errors from well to well variation of low length path and short length path by providing a threshold minimum to avoid florescence background that low length path may generate. The fluorescence from the target and reference genes is normalized with the flourescence from equal amount of Rox added to each well. Machines that do not require rox normalization count the flourescent signals using multiple optics over each well or optics that calculate florescence at each well at equal light path before entering the optics to sensors

Hope I am not arriving too late to the discussion, but I was wondering: do you need a different filter for the ROX dye or is it detected in the same channel as CYBRGreen? Mathew A Beale

You are late :) But yes , you do need different filters. Look up the wavelengths. i think Rox is n the red range.