In the Rotor-Gene, all channels can be used for independent multiplexing. This is in contrast to other real-time systems that require one channel be reserved for signal normalization (using the ROX™ passive reference dye, for example).
In addition, "virtual" channels can be created by mix-n-matching the excitation and emission frequencies. A virtual channel can be assigned a custom channel name and saved for future use (download the PDF file below "7-plex quantitation on the Rotor-Gene 6000" to see an example virtual channel application).
An example of a six-plex assay for SNP genotyping by quenched FRET probes is detailed here. Dyes spanning the entire spectrum, from UV (Blue channel) to infra-red (Crimson channel) were used. All primers and probes were combined in the one reaction. Amplification followed by melt analysis was done using a six channel Rotor-Gene (model 6600) and standard software.
Melt analyses show plotted results for five individuals (Fig 1). Each allele produced a distinct melt peak. Single peaks correspond to homozygous samples while two overlapping peaks (of about half-height at each allele location) indicate heterozygotes. Genotypes were automatically assigned by the Rotor-Gene software (to view individual analyses click on Fig 1 plots). Genotypes are summarized in Table 1 (right).
This assay is currently under development for forensic use and was kindly provided by Janice Nicklas, Ph.D. VT Forensic Laboratory, Dept. of Public Safety, 103 S. Main St., Waterbury, VT 05671, USA.
Resources
NEW! 7-plex Quantitation on the Rotor-Gene 6000 (706kb)
6-colour Multiplexing on the Rotor-Gene 6000 (550kb PDF)
Fig. 1: Six-plex SNP melt plots for a forensic typing assay. Derivative melt plots show peaks corresponding to each allele. Click each overhead tab to see the data for each channel. Channel name and the dye used are indicated on each plot (top right). Click each plot to see a detailed Rotor-Gene software analysis for that channel and the auto-call genotyping results obtained.
Table 1: Summary of genotyping data for a six-plex forensic typing assay. Genotypes of each individual tested are shown for all six SNP markers used by the assay.
