Two-Fold Quantification Discrimination of a 2-fold difference in starting copy number from an amplification reaction is a good measure of real-time performance. This is because a 2-fold change equates to a 1-cycle difference in the reaction, assuming 100% cycle efficiency (i.e. doubling of target at each cycle). Shown opposite is data that any real-time system would find hard to produce. We simultaneously challenged the Rotor-Gene 6000 with: ·    2-fold discrimination    (i.e. discrimination of 1 amplification cycle) ·    10 separate serial 2-fold dilutions, each in triplicate ·    Single-copy gene target amplification from a whole     human genome ·    Fast cycling (40 cycles in about 46 minutes) ·    Low probe concentration (about one quarter) ·    Standard commercial master-mix chemistry Notice how tight the replicates are, amplified in a third of the time and using a fraction of costly probe. And all achieved with standard chemistry.

Two-fold quantification on the Rotor-Gene 6000: BCL-2 human gene target (68 bp amplicon) amplified from total genomic DNA template (Promega Corp., Madison WI). Two-fold dilutions shown in triplicate, from ~256,000 copies (920 ng) down to ~500 copies (1.8 ng) assuming 3.59 pg/haploid genome. Primer concentration 300 nM, dual-labelled probe 60 nM, 40 cycle amplification completed in about 46 min using standard Platimum® qPCR SuperMix-UDG commercial master mix (Invitrogen Corp., Carlsbad, CA). Semi-log amplification plots shown of normalized fluorescence vs. cycle number with no smoothing applied and without ROXTM normalization.