Reproducibility and ROX™ The well-to-well uniformity of the Rotor-Gene means excellent experimental reproducibility, regardless of the number of replicates used or where they are placed in the rotor. It also means you don’t require a normalization dye such as ROX™ to achieve excellent results. However, the use of a normalization dye is an option on the Rotor-Gene 6000. Typical Results The experimental results shown opposite and summarised below illustrate the reproducibility of the Rotor-Gene 6000 and that ROX normalization has no discernable benefit. Without ROX Normalization: Average CT = 24.94 CT std. dev. = 0.05 Replicate CT (95% C.I.)= 24.93-24.95 With ROX Normalization: Average CT = 25.88 CT std. dev. = 0.06 Replicate CT (95% C.I.)= 25.86-25.89 Note that the small variation in final CT value observed across a full rotor of 72 replicates includes variation contributed by the CAS-1200 liquid handling robot used to set-up the reactions. As expected, pipetting variability was low due to the high-precision of the CAS-1200 robot. Note that there were no outliers in this experiment (all 72 well positions were used for the analysis). In summary, the results underline the excellent reproducibility typical of the Rotor-Gene 6000.

Reproducibility of amplification plots on the Rotor-Gene 6000 with or without ROX normalization. Amplification of bcl-2 gene from human genomic DNA (18.7 µg/mL, Promega, USA) using dual labelled probe chemistry. Each of the 72 replicates contained 1X buffer, 3 mM MgCl2, 0.2 mM dNTPs, 300 nM forward and reverse primer, 120 nM FAM-BHQ1 probe, 1.25 U Taq polymerase, 0.5 µL ROX and 1 µL of DNA. Each 25 µL reaction was aliquoted into 0.1 mL strip tubes from a master mix using the CAS-1200 robotic pipetting system (Corbett Robotics). The reaction was run using the dual-labelled Probe profile from the Advanced Wizard in the Rotor-Gene 6000 version 1.7 software. The profile includes a 2 min hold, followed by 40 cycles of 95°C for 10 sec and 60°C for 45 sec. Fluorescence was acquired on both the Green and Orange channels. Autogain optimization (built-in automatic sensitivity control) was carried out at the first acquisition temperature (5-10 starting fluorescence units).