Use of ROX as reporter dye on the LightCycler® 96 System

Technical TipFebruary 2013<strong>Use</strong> <strong>of</strong> <strong>ROX</strong><strong>as</strong> <strong>reporter</strong> <strong>dye</strong> on theLightCycler ® 96 SystemThe LightCycler ® 96 System supports seven <strong>dye</strong>s for multicolorapplications using Hydrolysis Probes:J Channel 1 (470/514): FAMJ Channel 2 (533/572): VIC, Hex, Yellow555J Channel 3 (577/620): Red 610, Tex<strong>as</strong> RedJ Channel 4 (645/697): Cy5<strong>ROX</strong> <strong>dye</strong> h<strong>as</strong> a similar excitation and emission spectrum <strong>as</strong> Red610 and Tex<strong>as</strong> Red, and therefore can be used in place <strong>of</strong> Red610 or Tex<strong>as</strong> Red in LightCycler ® 96 multicolor applications. Thefollowing observations are made:1. <strong>ROX</strong> is detected with sufficient sensitivity in Channel 3(577/620).2. The generic color compensation <strong>of</strong> the LightCycler ® 96s<strong>of</strong>tware compensates <strong>ROX</strong> quantitatively in multicolorapplications, showing no visible remaining crosstalk.When using <strong>ROX</strong> in the LightCycler ® 96 System, select Red610 in the Detection Format dialog box. In all instances <strong>of</strong> thes<strong>of</strong>tware (sample editor, raw data, analysis modules), <strong>ROX</strong> data(e.g., amplification curves, heat map values, result table values)are then displayed in the instance marked <strong>as</strong> Red 610.The enclosed data show a typical 4-color (4-plex) <strong>as</strong>say with thefollowing <strong>dye</strong> combination:FAM + VIC + <strong>ROX</strong> + Cy5The data show various target combinations including <strong>ROX</strong>amplification only (see Figure 1), <strong>ROX</strong> amplification withall dual-color combinations (see Figures 2 – 4), all possibletriple color combinations (see Figures 5 – 8), and a 4-colorcombination (see Figure 9).

In order to show the absence <strong>of</strong> crosstalk in multicolor <strong>as</strong>says, a quatroplex <strong>as</strong>say w<strong>as</strong> performed.Figure 1Figure 2<strong>ROX</strong> amplification only.<strong>ROX</strong> amplification with FAM dual-color combination.Figure 3Figure 4<strong>ROX</strong> amplification with VIC dual-color combination.<strong>ROX</strong> amplification with CY5 dual-color combination.Figure 5Figure 6<strong>ROX</strong> amplification with FAM, VIC triple color combination.<strong>ROX</strong> amplification with VIC, CY5 triple color combination.2

Figure 7Figure 8<strong>ROX</strong> amplification with FAM, CY5 triple color combination.FAM amplification with VIC, CY5 triple color combination.Figure 9As shown, in channels with non-amplified targets, no remainingcrosstalk is visible.RemarksJ Note that multiplex amplification requires optimization.J <strong>ROX</strong> is used <strong>as</strong> <strong>reporter</strong> <strong>dye</strong> in the listed application and doesnot function <strong>as</strong> a reference <strong>dye</strong>.J The LightCycler ® 96 System does not require or support <strong>ROX</strong> <strong>as</strong>a reference <strong>dye</strong> in M<strong>as</strong>ter Mixes.<strong>ROX</strong> amplification; 4-color combination with FAM, VIC, and CY5.The experiment w<strong>as</strong> performed using the RocheF<strong>as</strong>tStart Essential DNA Probes M<strong>as</strong>ter,Catalog Number 06 402 682 001.www.lightcycler96.comFor life science research only.Not for use in diagnostic procedures.FASTSTART and LIGHTCYCLER are trademarks <strong>of</strong> Roche.All other product names and trademarks are the property <strong>of</strong> theirrespective owners.For patent license limitations for individual products, ple<strong>as</strong>e refer to:www.technical-support.roche.comPublished byRoche Diagnostics GmbHSandh<strong>of</strong>er Straße 11668305 MannheimGermany© 2013 Roche Diagnostics.All rights reserved.