1. Next-Generation Performance and More Capabilities
Introducing the
Next-Generation Performance and More Capabilities
The aim of this slide set is to introduce the Rotor-Gene 6000 and the advantages of the rotary reaction format.
Slides are annotated so please check the notes here for further explanation as you go.
corbett
LIFE SCIENCE

2. What is the Rotor-Gene 6000? An integrated device that:
Maintains identical well-to-well conditions to monitor micro reaction tubes
Illuminates and collects a wide range of optical signals
Enables exquisite control of thermal conditions
Provides an open platform for all chemistries
Features a very fast data acquisition rate
Capable of the broadest application set:
Real-time analysis (e.g. quantitative amplification)
End-point analysis (e.g. SNP genotyping)
SYBR melt analysis
HRM (high resolution melt) analysis
Concentration analysis
+ future applications…
Critically important to real-time analysis (and the main strength of the Rotor-Gene) is its ability to effortlessly maintain identical thermal and optical conditions to every reaction tube.
Its design allows a wide range of optical signals to be used, from infra-red to ultra-violet, the widest of any system.
Thermal control and uniformity is unparalleled, as we’ll show in later slides,
Importantly, our philosophy is to provide a great open platform for use with any chemistry and application. Because we do not manufacture or focus on any one particular chemistry, we don’t try and limit the usefulness of the instrument, as other suppliers do to suit their own reagent products.
Finally, the speed with which the Rotor-Gene can acquire data opens up new application possibilities such as HRM.
All this functionality makes the Rotor-Gene capable of the broadest real-time application set available.
There are other instruments that can do the first three on this shortlist, but only the Rotor-Gene can also do HRM and concentration analysis, for example.

3. How does the rotary format work?
400 RPM
Samples spin continually during a run  400 RPM (heating or cooling)
High-speed data collection  all samples read in one revolution (0.15 sec)
G-force keeps reagent at base of tube  removes bubbles & condensation  will not pellet components
Continuous movement means no variation  well-to-well thermal or optical
>10 G
What makes the Rotor-Gene different is its centrifugal rotary design. So lets now examine how this works.
A rotary design is distinct from the standards 96-well “block” designs and even from other circular formats such as the carousel design (like that used in the LightCycler, for example). A centrifugal rotary instrument spins quickly in comparison to a carousel and doesn’t need to pause at each tube to collect data as other systems do.
Click 1: Speed is maintained at 400 rpm throughout the run for both data collection and heating/cooling phases.
Click 2: At 400 revolutions per minute (rpm) all the tubes pass the optical detector in 0.15 seconds, allowing data points to be captured very quickly and from all phases of the protocol.
Click 3: The speed of the rotor imparts over 10G of down force on the reaction. This is not enough to affect normal reaction kinetics, but it does spin-down any air bubbles introduced by pipetting and any condensation droplets are also centrifuged back into solution.
Click 4: Again it is clear that fast moving tubes spinning in actively moving air and passing the same optical detector suffer none of the well-to-well variations that other designs do (particularly block-based instruments).

4. Many advantages over other designs including:
Why rotary?
Many advantages over other designs including:
All wells are iso-thermal
All wells are iso-illuminated
Super fast data acquisition rate
Minimal maintenance and calibration
Simple and automated verification testing
Enables the broadest application set
We first developed a 96-well real-time instrument similar to other manufacturers, but this was quickly rejected as having limited capability.
Why did we totally rethink and revise the real-time format?
Because we quickly realized that a rotary design offers many advantages, including… (bullet points)

5. 3rd Generation Rotary Design The Rotor-Gene 6000 is based on proven technology
2003
Rotor-Gene 6000
2006
It’s important to realize, that although the rotary format is different, it is a tried-and-tested concept.
Corbett Life Science has constantly refined and developed the rotary format for real-time analysis over many years .
Both the hardware and software are now in a very advanced stage of development.
With the Rotor-Gene 6000 we have a 3rd generation product, and the software for instrument control and analysis is in its 7th major revision.
So while the 6000 series is new, it rests on many years of successful product development and worldwide sales.
Rotor-Gene 2000 instruments have now seen years of constant use with most of the original units still active today.
Rotor-Gene 2000
2000

6. The Rotor-Gene was recently honored with the prestigious Frost & Sullivan technology innovation award in the USA

7. Optics
Let’s first look at the optic system and how it works.

8. Cross-section of rotary optics
Reaction Chamber
Detection Filters
Lens
PMT Detector
Assembly
Spindle/Motor Assembly
Tubes in Rotor
Spin Past Optics
LED Light Source Assembly
To better understand centrifugal design, let’s look at an x-ray cross section view through the middle of the RG.
Click 1: Firstly, here’s the reaction chamber, a light-sealed unit, the top half of which swings open with the RG lid.
Click 2: Now here’s the rotor in position (here colored red). This is a 36-well rotor holding the largest tube type (0.2 mL). This makes it easier to see the individual tubes.
Click 3: This allows the tubes to spin around and pass the optic subsystem, which we’ll look in a moment.
Click 4: The rotor doesn’t hang in mid air as shown, it sits on a motorized spindle assembly, just like in a centrifuge.
Click 5: The light source is an array of individual LEDs (light-emitting diodes) arranges in a “gattler” assembly. The gattler turns to allow individual LEDs to be used as appropriate. There are up to 6 LEDs depending on the model.
Click 6: Detection of optical signals is via a photomultiplier (PMT), positioned at the side of the unit.
Click 7: The Detection subsystem contains a lens
Click 8: And a set of up to 6 filters, depending on the model.
Click 9: To detect signals, the selected LED illuminates each reaction as it spins past with a high-power burst of light.
Click 10: The reaction emits fluorescent energy…
Click 11: …which is focussed through the lens, passes through the selected filter and is detected by the PMT and reported.
It’s important to note that each tube is moved to the identical optical pathway. This simply means that there is no optical variation at each tube position. This is why the RG does not require any optical callibration, as other systems do.
Click 12: The RG is heated and cooled by fast-moving air. Even when collecting fluorescent signals the air in the chamber is circulating around each tube as they spin past the detector.
This ensures well-to-well thermal variation is absolutely minimal—below detectable levels in fact. Contrast this with other systems that typically suffer one degree or more difference across the block, particularly in edge wells.
To complete our understanding of the mechanism, vents are opened and cool air is imported through the base of the chamber and hot air is expelled through the top of the chamber (and out the vents on the lid) when the instrument is in cooling mode (not shown)
When heating, the chamber seals and an overhead heating element (not shown) raises the temperature of circulating air.

9. Rotary optics 3D animation
rotor spins tubes at 400 rpm
filter set
(rotates for each channel)
sensitive PMT (photomultiplier) detector
lens
Here is a simple 3D animation to help explain the basics of rotary design.
LED light source
(rotates for each channel)

10. Rotary Design means: low maintenance & maximum convenience
No optical normalization needed (thus no special reagents either)
No passive reference dye needed (e.g. ROX™)
No lamp replacement or maintenance (lifetime guarantee on lightsource)
No block to clean (because tubes hang in mid air)
No bubbles in reaction (automatically removed by centrifugal rotor)
Tube caps can be labeled (not possible with 96-well systems)
Supports several tube formats (by simply swapping rotors)
From a day-to-day running perspective (particularly a shared laboratory environment with many casual users), the Rotor-Gene offers some very tangible benefits, including…
(listed)
Note: ROX is the name of a fluorescent dye used for normalizing well-to-well optical variation. It’s used as a housekeeping dye or “passive reference”. The signal from the ROX dye is collected at each cycle along with the reporter dye. The ROX signal is not part of the amplification, so it’s considered “Passive”. For normalization, the reporter dye signal is divided by the ROX dye signal. Wells that are bright get divided by a larger ROX value than wells that are more dimly lit. In this way the final data signal is normalized.
ROX was pioneered by Applied Biosystems but it or an equivalent is added or optionally added to many commercial master mix reagents. The Rotor-Gene supports passive reference normalization but the results normally show no discernable improvement, as we’ll see in later slides.

11. Light-Emitting Diode (LED) Light Source
LEDs last >100,000 hrs
Low-power, low-temperature device
LEDs have a focussed light-emitting area
Separate LED used for each channel from infra-red (IR) to ultra-violet (UV)
Estimated lifespan ~40 years (assuming 4 runs every working day)
Rotor-Gene LEDs are guaranteed for the life of the instrument!
LEDs are an ideal excitation source for real-time analysis compared to lamps and lasers used in other systems because…

12. 5 Models to suit different needs and budgets:
Channel Excite/Detect (nm) Example fluorophores detected
Blue 365/460 BiosearchBlue™, Marina Blue®, Bothell Blue®, Alexa Fluor® 350
Green 470/510 FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488
Yellow 530/555 JOE™, VIC™, HEX™, TET™, Yakima Yellow®, Cal Flour® Gold 540
Orange 585/610 ROX™, Cy3.5®, Texas Red®, Alexa Fluor® 568, CAL Fluor™ Red 610
Red 625/660 Cy5®, Quasar 670™, LightCycler Red 640®, Alexa Fluor™ 633
Crimson 680/712 LP Quasar705™, LightCycler Red 705®, Alexa Fluor® 680
HRM 460/510 SYTO®9, LC Green®, LC Green™Plus+, EvaGreen™ UV IR
5 Models to suit different needs and budgets:
Plex Green/Yellow
Plex HRM Green/Yellow + High Resolution Melt channel
Plex Green/Yellow/Orange/Red/Crimson
Plex HRM Green/Yellow/Orange/Red/Crimson + High Resolution Melt channel
Plex Blue/Green/Yellow/Orange/Red/Crimson
The RG6000 has 6 channels that can be configured from the seven available options detailed here.
Note some standard dyes for each channel are listed.
(Note LP=long pass; this means all wavelengths longer than 712 nm are detected. Other channels are band pass, typically +/- about 10 nm)
Click 1: In fact there are 5 different models available:
Click 2: A simple 2-plex instrument configured for standard chemistries such as SYBR melt analysis and diplex TaqMan reactions (using FAM/VIC dyes for example)
Click 3: A 2-plex instrument with the exciting new HRM capability (more on that later)
Click 4: A 5 plex model that uses the entire range of standard dyes for higher levels of multiplexing
Click 5: A 5 plex model with HRM
Click 6: and finally a 6-plex model that adds a UV channel to embrace emerging technologies.
Click 7: Note that we use a “Plex” designation for channels since all channels can be used to monitor reporter fluorophores. We do not need to reserve a special channel for a normalization or reference dye as other systems do.
NOTE: ROX™ normalization is not needed so all channels can be “plexed” for separate reactions

13. Thermal Control
Now let’s turn our attention to the impact of temperature on an amplification reaction…

14. Thermal Precision (uniformity) in a Block-based Cycler
± 0.50 ºC (or more) across the 96 well block (thus > 1.00 ºC variation is typical)
Corner and Edge wells most affected
NOTE: Fluorescence  1/Temp
Localized “hotspots” as Peltier device junctions begin to fail
The Rotor-Gene does not use Peltier devices—because they fail unpredictably and are expensive to repair
Most real-time systems have adapted an optics subsystem onto a standard 96-well or 384-well thermal cycler.
Click 1: Unfortunately however, temperature variation is unavoidable across a metal 96-well block. In fact, well-to-well variability is the most challenging but important issue affecting real-time instrument performance.
Click2: Note the Rotor-Gene thermal uniformity and resolution are unparalleled.
The images are of a color-enhanced thermal contour map of a typical metal 96-well block heated and cooled using Peltier devices surface-mounted to the underside (shown in black). The gradient is from red (hot) to blue (cool).
Click 3: Dissipation of heat from the Peltier devices is not uniform across the block (”edge effect”) or vertically from the base to the top of each sample well. In addition, localized hot-spots can also occur (as shown) when individual Peltier device junctions begin to fail.
Click 4: Fluorescent data signals are also affected by thermal variation since fluorescence is inversely proportional to temp (i.e. 1/Temp).
Click 5: In addition, most real-time systems use Peltier devices to actively heat and cool samples. The Rotor-Gene doesn’t use these devices, a distinct advantage because, like light bulbs, Peltiers fail unpredictably and are expensive to repair. They can also produce localized “hotspots” as device junctions inevitably begin to fail.
NOTE: Thermal variability is further exacerbated by the new trend to speed-up cycling times. Increasing speed worsens thermal performance because the faster a block is heated or cooled, the greater the well-to-well thermal variation observed.
(FYI: The hotter region at the top edge of the sample wells originates from heat transferred from a typical “heated lid” mechanism used to help curb sample condensation effects. The Rotor-Gene uses centrifugal force to continually remove trace sample condensation.)
NOTE Rotor-Gene 6000 specifications: Uniformity: ±0.01°C, Resolution: ±0.02°C

15. Heating mechanism Heater elements switch on
Centrifugal fan drives air around chamber
Note: holes in the rotor allow free airflow
Chamber vent seals to contain air
A longitudinal cross section (front to back) through a Rotor-Gene 6000 is shown here.
You can see the location of the tubes in the rotor which sits in the middle of the reaction chamber on top of the motor spindle assembly.
Click 1: When the heating mechanism switches on, the vent to the rear of the Rotor-Gene seals to contain the air in the chamber.
Click 2: Heater elements switch on and…
Click 3: A centrifugal fan drives the heated air around the chamber
Click 4: Air can circulate freely around the reaction chamber through holes in the rotor (red areas)

16. Cooling mechanism Heater elements switch off
Centrifugal fan drives air around chamber
Chamber vent opens expelling hot air
Note: holes in the rotor allow free airflow
Centrifugal fan Drives air into chamber
The Rotor-Gene 6000 cools the samples as follows:
Click 1: The heater elements turn off
Click 2: Cool air is ducted in from underneath the unit
Click 3: A centrifugal fan forces the cool air into the reaction chamber
Click 4: The rear vent opens to expel the hot air
Click 5: The chamber cools rapidly
Cool air in

17. Thermal Equilibration: Rotor vs. Block
72.5 Degrees
72 Degrees +/- 0.01
71.5 Degrees
96 ºC
72 ºC
60 ºC
Here we’ve co-plotted the difference in individual well temperatures vs. time for representative wells in a block cycler and a Rotor-Gene.
Notice how temperature in the Rotor-Gene (red plot line) remains iso-thermal (equal) between wells DURING programmed temperature transitions.
Conversely, the temperatures vary between wells in the block system (blue plot lines).
Note also how an extra equilibration time is needed before all wells in the block system reach the same set temperature.
Because an equilibration time is not needed in the Rotor-Gene, programmed cycle times can normally be reduced by about 15 seconds at each set temperature without affecting results. This alone can significantly shorten experimental run times.
So in the Rotor-Gene every tube changes temperature at the same rate. This negates another well-to-well variable normally affecting real-time reaction kinetics.
Equilibration time in Rotor-Gene is 0 sec (for ± 0.01 °C)
Equilibration time in 96 well block is 15 sec (for ± 0.5 °C)
Up to 50% faster run times with better uniformity between samples
TIME

18. Thermal Accuracy Instrument Calibration and Performance Verification
We have seen how well-to-well thermal uniformity is crucial.
We have also seen how thermal equilibration tie uniformity is also important.
These are factors affecting temperature precision, i.e. how reproducible the temperature is.
What we haven’t covered is thermal accuracy. Thermal accuracy is how close the temperature in °C reported by the instrument is to the to the absolutely correct temperature.
The only solution to this is proper thermal calibration and ongoing verification that the calibrated values are correct.
This is an area of critical importance to the reproducibility and transportability of protocols between laboratories and across the world.
For this reason we have developed the most advanced portable solution to routine accuracy verification; the OTV system.

19. For Checking and Calibrating Thermal Accuracy and Optical Performance
Increasingly, laboratories require routine verification and validation of instrument performance and thermal accuracy
The OTV Kit automates routine verification testing on the Rotor-Gene
The OTV system comprises an OTV Disc, optical insert accessories and a CD
With the kit, verification of instrument performance and thermal accuracy can be done at up to 30 times within the 6 month expiry date of the rotor consumable
Automated verification testing in this manner is unique to the Rotor-Gene
This slide was designed to be self explanatory

20. OTV Mechanism
The OTV system* uses the chemical properties of three different thermochromic liquid crystals (TLCs) as an absolute temperature reference.
When heated, a TLC changes from opaque to transparent at a very precise temperature. But because TLCs do not fluoresce, a fluorescence scatter plate is inserted over the optics to enable detection by the Rotor-Gene.
The Rotor-Gene measures the precise temperature transition of each TLC. This reported value is compared to the known calibrated value to verify the instrument is within specification. If not within specification, automatic re-calibration of the Rotor-Gene can be done at the press of a button.
OTV disc in rotor
Rotor-Gene 6000 Scatter Plate Insert
This slide was designed to be self explanatory
Rotor-Gene 3000 Scatter Plate Insert
*patent pending

21. OTV Report
The HTML report file can be saved, printed, ed or exported to MS Word as a record of the verification test
The report indicates the instrument was within specification when “No Adjustment Required” is stated (as shown here)
Detailed analysis data is also reported
This slide was designed to be self explanatory

22. Software More analysis options Raw data export
Standard Curve Quantitation
2 Standard Curves Relative Quantitation
Delta Delta Ct Relative Quantitation
Comparative Quantitation
Relative Expression Software Tool (REST)
LinReg (Assumption-Free Analysis)
Melt Analysis
High Resolution Melt Analysis
End-Point Analysis
Allelic Discrimination
Scatter Graph Analysis
Concentration Analysis
More analysis options
Raw data export
For 3rd party software analysis
Unlimited user software license
No additional license fees
All users can copy & run the software to analyze files remotely
Upgrades free (by web download)
The Rotor-Gene 6000 software gives you more real-time data analysis options as standard, including support for many you won’t find elsewhere, such as:
- REST (relative expression software tool) - LinReg (Linear Regression/Assumption-Free Analysis) - Comparative Quantification
In addition, you can determine the amplification efficiency of individual reactions or export the raw data for outside analysis.
Unlike many other systems, the Rotor-Gene comes with an unlimited user software license, which means it can be installed and run as often as required to suit all system users.
The software is always being improved with new capabilities, and new upgrades are free by web download.

23. Plug-and-Play Portability
Robust design suits transportability
No optical alignment or calibration needed
Self-configuring USB connection to computer
Easy to carry
Small: 370 mm (14.6") W, 420 mm (16.5") D, 275 mm (10.8") H
Light: 14 kg (31 lbs)
The Rotary design is not as delicate as other systems. If you need to move the instrument between labs or even put it in the back of your car to take it across town you can. You don’t need o re-align optics or perform any recalibration afterwards.
The instrument also connects automatically to the control computer.
It’s also small and light compared to other systems (details indicated on slide).

24. Tube Formats and Sample Handling
The Rotor-Gene can use a variety tube formats compatible with different sample handling and workflow requirements.

25. Tube Formats 2. 1. 36  0.2 mL PCR tubes Attached flat or domed caps
NOTE: optical caps are not required since detection is through the base of the tube 2.
72  0.1 mL tubes - allow small reaction volumes (5–10 µL) - in strips of 4 for ease of use
- Frosted cap extensions allow write-on labelling + easy handling
Here are the two individual tube formats supported;
Standard 0.2 mL PCR tubes and
Click 1: mL strip tubes.
Some users prefer to separate the strip tubes into individual tubes that can be capped immediately after reaction set-up.
The larger 0.2 mL tubes can be useful when large-volume reactions are needed (for example, ≥100 µL reaction to detect very low-abundance targets)

26. Gene-Disc™ Plates 3.
Gene-Disc™ mL tubes in a rotary “plate” design - Tubes oriented vertically (not angled)
- Manual or automated loading
- Heat-sealed in seconds 4.
Gene-Disc™ For 96 sample workflow + 4 extra controls
30 µL wells in a rotary “plate” design
- Manual or automated loading - Wells oriented vertically (not angled)
- Heat-sealed in seconds
There are two “plate” equivalent tube formats for the Rotor-Gene: the Gene-Disc 72 and
Click 1: the new Gene-Disc 100.
These formats suit high volume or robotic workflow situations…

27. Direct Robotic Setup in Gene-Disc™ Plates
Vertical tube orientation means a robot can set up all reactions directly into Gene-Disc tubes
The new heat sealer provides a permanent or removable film seal (user selectable switch)
Gene-Disc rotor ready for cycling
The vertical orientation of the tubes in the Disc allows a robotic tip to access every well (in non-Gene-Disc tube formats the tubes are angled making so direct set-up of reactions in a rotor is not possible).
Click 1: Here we can see robotic reaction set-up on a CAS-1200 workstation (Corbett Robotics).
Click 2: After set-up, plates are heat-sealed then…
Click 3: …transferred to the rotor for cycling.

28. Amplification Performance
Let’s now look at some example data obtained on the Rotor-Gene 6000…

29. Replicates (full 72-well rotor)
No ROX normalization
With optional ROX normalization
Here we examine a full rotor of replicate reactions.
Amplification plots are semi-log scale.
Details on the run are below.
Note:
1. there are no outliers—all reactions are shown and worked equally well.
2. Replicates are very tight both with and without ROX passive reference normalization. This underlines our claim for both thermal and optical well-to-well uniformity in a rotary instrument.
3. Click 1+2: note the standard deviation and range of Cts for a 95% confidence interval (i.e. if you repeated the experiment 100 times, 95% of Cts would fall in this range).
AIM:
Determine if ROX normalisation can improve replicates in a full rotor run.
METHOD
Amplification of the bcl-2 gene from human genomic DNA (18.7 ug/mL, Promega, USA) using dual labelled hydrolysis 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 uL ROX and 1 uL of DNA. Each 25 uL reaction was aliquoted into 0.1 mL strip tubes from a master mix using the CAS1200N robotic pipetting system(Corbett Robotics).
The reaction was run using the Hydrolysis Probe profile from the Advanced Wizard in the RG6000 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. An Autogain optimisation was carried out at the first acquisition temperature ( Fl units).
ROX normalisation was achieved by selecting the Options tab on the Green channel raw data and selecting Normalise to Orange channel. The normalised channel was analysed using the Quantitate analysis option. A threshold was set manually to a value of 0.1. The standard deviation and Ct mean were recorded and compared to the values obtained from the Green channel analysed data using the same threshold.
RESULTS
The results demonstrate no significant difference in the standard deviaiton between ROX normalised data and non normalised data (0.06 vs. 0.05). The mean Ct value for the ROX normalised data was 1 cycle higher compared with the non normalised data (25.88 vs ) using the identical standard threshold setting (0.1).
From the results we can conclude that ROX normalisation has no significant measurable benifit to real time analysis data obtained on the RG6000. This underlines the well-to-well uniformity of the Rotor-Gene system. Note that there are no outliers (all well positions are used for the analysis).
The variation in final Ct value observed across the entire rotor and all replicates includes variation from pipetting error contributed by the CAS-1200N robot. As expected, pipetting variability is low due to the high-precision nature of the CAS-1200N.

30. 2-fold quantitative discrimination
2-fold discrimination (=1 PCR cycle)
10 separate dilutions in triplicate
No ROX normalization
No data “smoothing”
single-copy gene amplified from whole human genomic template
Fast cycling (40 cycles, 46 min)
Standard commercial master-mix
Low probe (60 nM = ¼ dilution)
256,000 copies
500 copies
Here are example results for quantitative real-time amplification on the RG6000.
The semi-log amplification plots pictured show a 2-fold dilution series from 256,000 copies down to 500 copies, done in triplicate. If you look carefully down in the very low signal/noise area you can just see the separate replicate traces. However, by the time plots reach the threshold the replicates overlay and can’t be discriminated. This is because the replicates are virtually identical, which is ideal. A 2-fold dilution series like this is demanding because it must clearly discriminate a single PCR cycle. You can clearly see the results are indeed exactly one cycle apart over the entire series of 10 dilutions!
Click 1: A series of 10 separate dilutions, each in triplicate is shown.
Click 2: Note also that no normalization to a reference dye (such as ROX) was done to achieve this result. You can apply ROX normalization if you wish, but it is not normally used and certainly not necessary as you can see. This is due to the superior rotary design format we use.
Click 3: Note the “jaggy” lines in the lower noise area, this indicates that no smoothing adjustment has been made to the curves at all. What you see is literal cycle-by-cycle readings connected on the graph. All any real-time system can do is collect a single consolidated data point per cycle, so this view is the most technically correct. Most software interpolates between successive data points to smooth the curve visually—this is also an option on the Rotor-Gene.
Click 4: These results are from dual-label probe assays targeting the human BCL-2 gene, a single copy gene in the genome. The amplified template for this experiment was whole human genomic DNA and not a less complex cloned target source (such as a plasmid).
Click 5: Results were generated using fast cycling; the 40 PCR cycles you see here were completed in about 40 minutes total.
Click 6: Usually fast PCR cycling requires specialized and more expensive chemistry. These results were obtained with a standard commercial master mix (see last line of Fig legend for details).
Click 7: The sensitivity of the standard RG6000 optics allow you to use lower probe concentrations than in other systems. In this case we diluted the standard probe concentration of 250 nM down to 60 nM; approximately a 4-fold dilution. Further dilution also works well. This means that your expensive probe can stretch to many more experiments, saving substantially on running costs.
Taken together you can see this was a challenging experiment in several respects, however the results are exemplary!
It’s rare to see 2-fold data of this quality, even without the challenging conditions we set up here.
BCL-2 human gene target (68 bp amplicon) amplified from total genomic DNA template. Semi-log amplification plots shown of normalized fluorescence vs. cycle number with no smoothing applied and without ROX™ normalization. Primer concentration 300 nM, dual-labeled probe 60 nM, 40 cycle amplification completed in 46 min using standard Platimum® qPCR SuperMix-UDG commercial master mix (Invitrogen Corp., Carlsbad, CA).

31. Concentration Measurement
Concentration measurement is a new application for the Rotor-Gene and one that other real-time systems don’t support.

32. DNA Concentration Measurement
1000
900
800
700
600
500
400
300
200
100
The Rotor-Gene is fully equipped to do DNA concentration measurement using fluorescent dyes
Using a standard run protocol and integrated analysis software, the concentration of unknown samples is determined from a standard curve.
Fluorescence
No DNA controls
The RG is the first real-time instrument able to directly determine the starting concentration of nucleic acids using fluorescent dyes such as PicoGreen and RiboGreen.
On the graph you can see DNA standards from a commercial kit quantified using PicoGreen dye.
Click 1: Negative controls appear at the origin
Click 2: Replicate standards are shown from 100 up to 1000 pg/µL. The curve is drawn using the standard spline curve fit option of the RG software.
It is worth considering the future use of this capability: we believe the wide excitation and detection range and sensitivity of the RG6000 may allow for automatic normalization of gene expression to starting template. This would replace the traditional use of a housekeeping gene (endogenous control) for normalization purposes. To work, unknown samples would be prepared with both a probe and an intercalator dye. The two dyes must have non-overlapping spectra, so a green channel dye such as PicoGreen could be used with an infra-red (crimson channel) probe dye.
Providing the concentration of input template is within the range of detection as shown here, then gene expression results could potentially be normalized to the initial fluorescence level measured before amplification. To date however, we have not investigated this possibility in detail.
Concentration pg/µL
A DNA standard curve with replicates is shown. Curve interpolated using a spline curve fit (Rotor-Gene analysis software). Data was obtained using reagents in the Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen Corp., Carlsbad CA). Standard Rotor-Gene concentration analysis run protocol was used. 10 µL PicoGreen® (diluted1/200 in 1 TE buffer) was combined with 10 µL of each standard (diluted in 1 TE buffer). Final volume 20 µL.

33. SYBR Melt Analysis and HRM™ (high resolution melt) a new application for a new type of instrument
High Resolution Melt (HRM) is, as the name suggests, an extension to the melting analyses done previously with SYBR Green I dye. HRM creates new analysis opportunities but requires next-generation instrumentation to work well. We are pleased to say that HRM is fully supported on the RG6000 platform.
HRM enables very precise melt analysis that can discriminate even single base changes in amplification products.

34. SYBR™ Green I Generic dsDNA intercalation dye Inexpensive & simple
Used for real-time PCR product detection
Used for DNA dissociation (melt) analysis
Widely used
To date, the standard dye for melt analysis has been SYBR Green 1.
It’s a “Generic” fluorophore, meaning that it will detect any double-stranded DNA (dsDNA) product by intercalating into the minor groove of a DNA double-helix, as shown diagrammatically here (SYBR molecules in green)
The mechanism by which it fluoresces when attached to a DNA molecule is unknown, but it works very well and produces a bright signal when properly illuminated.
Being only a dye, it’s relatively inexpensive and simple to use, and has been used to monitor real-time amplification reactions for many years. The mechanism to monitor the amplification process is simple: as dsDNA product (amplicons) accumulate during amplification, more dye intercalates with them and the fluorescent signal increases.
An important second application is the characterization of amplified products by the way their particular double-stranded sequence dissociates into single strands with increasing temperature. This is called dissociation analysis but commonly referred to as a “melt” analysis.
Melt analyses characterize dsDNA products according to their particular sequence; the sequence length, or GC content. It’s a good compliment to gel analyses as we’ll see, which can characterize products by size only, and it’s now a widely used method.

35. SYBR melt analysis of a DNA fragment
Raw data plot
Fluorescence drops as DNA melts and SYBR is released
Derivative data plot
This “rate” curve peaks at maximum dissociation rate which is indicative of the Tm (temperature of melting)
Here we see SYBR Green 1 melting analysis on a single PCR product.
The topmost graph plots raw fluorescent signal against increasing temperature on the x-axis (horizontal).
Notice how fluorescent signal decreases as temperature increases. This is because the dsDNA in the tube begins to disassociate (or “melt”) into single strands. Melting releases the dye and thus lowers the signal. Very simple.
Normally, the process reaches a critical point and the product begins to melt apart very quickly. You can see this as the steep part of the plot. The midpoint of this steep section (about 78°C for this example) is the point where 50% of the molecules have disassociated (melted). This is the definition of the Tm (the temperature of melting). So the Tm for this particular amplicon is about 78°C.
Now this type of data plot is not always easy to interpret, so for analysis a “derivative” plot is normally used which plots the rate of change of fluorescence vs. temperature. As you can see, the rate is initially slow, then increases, then slows again. This produces a “peak” in the derivative plot, the midpoint of which is the point of maximum rate of change in fluorescence and reflects the Tm.

36. SYBR™ melts can reflect product size
500 bp fragment
250 bp fragment
500 bp fragment
250 bp fragment
Melting analysis results reflect dsDNA dissociation behavior as strands separate with increasing temperature.
What makes one dsDNA different from another is its sequence. Here we can see the raw data and derivative data plots for two DNA fragments that differ in size.
As you might expect, the smaller fragment melted at a lower temperature. Thus its Tm is lower (by 2-3°C).
Note however, that clear differences in melt plots based on fragment size are not always so apparent, because the GC content and even the sequence of bases can have an even greater effect on the observed melt plots than size alone.
Raw data plot: fluorescence vs. temp.
Derivative data plot: dF/dT vs. temp

37. SYBR™ Green I melts can reflect sequence detection of alleles
Allele A
Allele A
Allele B
Allele B
Primer Dimer
Low primer conc (50 nM)
Single band contains two species (alleles)
High primer conc (900 nM)
Primer-dimer appears as a third species
Melt curves analysis is not the same as traditional gel electrophoresis analysis and provides different but complimentary data.
Here’s a comparison of an amplification product analyzed on a gel (below) and by derivative melt curve.
On the left you can see a single amplification product that produces two peaks on a derivative melt curve. But how can this be? The answer is that there are in fact two products present that are the same size. In fact they are alleles of the same amplified target and, in the heterozygous state here, produce products with only minor sequence differences that nevertheless amplify using the same primer set.
On the right, the primer concentration has been increased. This has produced a population of spurious products visible as a “smear” on the gel. It has also generated a clear primer-dimer artifact. Now the melt curve shows an additional third peak (in addition to the two allele peaks) corresponding to the primer-dimer Tm.

38. “Saturation” Hypothesis
SYBR™ Green I is toxic to PCR, so concentration used is very low
Some dye can relocate as melting begins
Theoretically, unsaturated binding may allow dye relocation during melts, making it less suitable for HRM
New dye technology for HRM Examples: EvaGreen, SYTO9
LC Green™ I
Dye saturation leaves no room for relocation events during melting
“Saturation” dyes are much less toxic, so concentration used can be higher
This may reduce dye relocation events and improve HRM results
SYBR® Green I
HRM technology has been driven by new developments in instrumentation and intercalating dye technology. New HRM dyes are referred to as “saturation” dyes. Lets examine what this means.
While not fully understood, the following hypothesis is currently used to explain how these new dyes work:
SYBR is a very useful dye, but relatively toxic to amplification reactions, so it’s used at a low concentration in the reaction. As such, the dye does not intercalate at every possible position in the DNA sample. In other words the DNA is “unsaturated”. When the DNA strands begin to melt, dye can jump back into regions of the helix that remain double stranded. This can make the fluorescent signal fuzzy and reduce resolution of a resulting melt curve.
New dyes like EvaGreen, SYTO9 and LC Green are much less toxic, so they can be used at much higher concentrations; high enough to saturate all the binding positions. So when the strand melts there is no room for the dye to relocate to another location. This makes the changing signal more definite and can provide clearer melt plots.

39. HRM on the Rotor-Gene 6000 To support HRM an instrument requires:
high-intensity + high sensitivity optics
high-speed data capture
very precise temperature control
extreme temperature resolution
To support multiple wells:
Superlative thermal and optical well-to-well uniformity
Fluorescence
The Rotor-Gene 6000 is uniquely able to support both thermal cycling and HRM because it incorporates:
High-intensity optics
High-speed virtually continuous data capture
Precise thermal control
And extreme temperature resolution.
The rotary design also provides the well-to-well thermal and optical uniformity essential for a multi-sample instrument.
The example HRM SNP data shows 10 replicates of each genotype. All are easily discernable and autocalled by the Rotor-Gene 6000 software. 82 83 84 85 86 87 88
Temperature (°C)
Example SNP genotyping using HRM analysis. ACTN3 (R577X) SNP genotypes (C—T).
Ten replicates each genotype are shown.
Fragment pre-amplified using a 40 cycle fast protocol (46 min).

40. HRM workflow on the Rotor-Gene 6000
0.2 mL tubes
Run PCR and HRM
Autocall genotypes Up to 100 at a time
0.1 mL tubes
Gene-Disc™ 72
Running a HRM analysis on the Rotor-Gene 6000, on the other hand, is much simpler.
You can choose from a variety of tube formats and sample capacities.
Click 1: Both amplification and the subsequent HRM are done in the Rotor-Gene 6000
Click 2: A dedicated HRM analysis module makes comparing multiple samples easy and can easily auto-call genotypes.
Gene-Disc™ 100
Choose preferred tube

41. HRM Applications
Some HRM applications currently under investigation include:
Mutation discovery
Screening for loss of heterozygosity
DNA fingerprinting
SNP genotyping
Characterization of haplotype blocks
DNA methylation analysis
DNA mapping
Species identification
Somatic acquired mutation ratios
HLA compatibility testing
Association (case/control) studies
Alleleic prevalence in a population
Identification of candidate predisposition genes
Although HRM is a new technology, there is great enthusiasm for it and may applications are already under active investigation. Because HRM can detect even single base alterations in an amplified product, it provides a new tool to find new mutations.
Other uses involve the detection of genetic variation across a wide range of applications, as indicated here.

42. Automation
With ever higher demands on cost-saving and high-throughput, robotic setup is becoming ever more popular. We have a low-cost high-precision robotic solution developed with real-time applications on the Rotor-Gene in mind.

43. Precision Robotic Pipetting Improves Results
The CAS-1200 is a very small personal liquid handling workstation which is ideal for real-time reaction setup. It is also designed for ease of use for the novice and to handle all the Rotor-Gene tube formats.
CAS-1200™
Precision Liquid Handling System
Rotor-Gene 6000™
Six Channel Multiplexing System

44. 10 µL reaction volume, 18 replicates
Rotor-Gene™ results
10 µL reaction volume, 18 replicates
At 10 µL, both robotic and hand pipetting are still equivalent.
This is a great result as very small reactions are prone to variability due mainly to pipetting eror.
Hand pipetting
CT std dev 0.12
CAS-1200 robot
CT std dev 0.10

45. 5 µL reaction volume, 18 replicates
Rotor-Gene™ results
5 µL reaction volume, 18 replicates
At 5 µL, the robotic pipetting shows a clear advantage in higher consistency over hand pipetting.
This tiny reaction volume is normally considered too small to be set up routinely by hand.
Remember, a robot makes fewer mistakes and doesn’t fatigue nor get distracted by telephone calls, for example. So it also saves money because reducing human error means fewer repeated experiments.
Hand pipetting
CT std dev 0.64
CAS-1200 robot
CT std dev 0.12

46. “Extraction-to-Reaction”
Robotic Workflow for Real-Time Analysis
At Corbett Life Science, we have a complete automated solution for all steps in real-time workflow, from automated nucleic acid extraction on the X-tractor Gene to…
Click 1: automated reaction set-up on the CAS-1200 right through to…
Click 2: Real-time processing and analysis on the Rotor-Gene.
Click 3: We call this our “Extraction to Reaction” solution.
“Extraction-to-Reaction”

47. Summary Real-Time analysis is an exact science with many variables
Through good design, the rotary format best minimizes these variables
Precise and reproducible data is easier to achieve with the Rotor-Gene
Future applications such as HRM and concentration measurement can only be achieved on the Rotor-Gene, underlining it’s superior design
Choose from more tube formats and data analysis options
Verification testing and calibration is automated by the OTV system
The robust design delivers minimum maintenance, lowest operating costs and maximum convenience
There are so many positive attributes of the Rotor-Gene system that it’s hard to consolidate into a brief list.
There are many features not mentioned here, including the wonderful experimental report files that the analysis software generates (which really write-up your lab book for you) or even the different security levels; from run file signatures to operator access privileges (administrator, Analyzer or Operator) etc.
But at the end of the day, what you really need is an instrument that you can rely on to do the best possible job. And that’s what the Rotor-Gene delivers.

48. colors You can choose one of two gloss metallic colors (no cost).
The intent is to make it look great on the outside to reflect our pride on the mechanics we have engineered inside.

49. All slides  2006 Corbett Life Science. All rights reserved
Offices
Brisbane Australia
Corbett Robotics Pty Ltd
42 McKechnie Drive
Eight Mile Plains, QLD 4113 T F
United Kingdom
Corbett Research UK Limited
Unit 296 Cambridge Science Park
Milton Road, Cambridge CB4 0WD
T +44 (0)
F +44 (0)
Sydney Australia
Corbett Research Pty Ltd
14 Hilly Street
Mortlake, NSW 2137
T (Toll free) T F
USA
Corbett Robotics Inc
185 Berry Street, Suite 5200
San Francisco, CA 94107
T (Toll free) T F
Web
All slides  2006 Corbett Life Science. All rights reserved

50. Conventional vs. Real-Time PCR
Conventional HIV PCR
Real-Time HIV PCR
Reverse
transcription
Reverse
transcription
and
PCR reaction
Quantitative
result
PCR
reaction
Transfer to
Nested PCR
reaction
Gel
electrophoresis
Southern
Blot
DNA
Sequencing
Densitometry/
Phosphor-
imaging
Manual or
automated
analysis
Real-Time PCR allows cost- and time-effective template quantification

51. Why is it Required? Viral load (CMV)
Quantification of bacteria (E.Coli)
Genetically modified organisms (Soy Bean)
Expression studies for pure research
Levels of expression through development
Comparison of diseased and normal cells or tissues
Expression studies for clinical applications
Measurement of genes coding for a drug resistance
Food testing
Additives
Contamination

52. PCR Dynamics Buffer changes (pH) Limiting factors Primers dNTPs
Thermostable enzyme
MgCl2
Primer binding properties
Length of product (eg SYBR Green)

53. Limitations of Non Real-Time Quantification
Assumptions on reaction consistency and uniformity
Assumptions on exponential region
Narrow dynamic range
Long optimisation and set up times
Long run and analysis times
High levels of inherent inaccuracy and variation

54. Advantages of Real-Time Analysis
Remove the “guess work”
Able to see in real-time the increase in a sample production
Check exponential part of the amplification
Broad dynamic range
As each sample is measured at its optimum point of amplification, a greater dynamic range can be measured
Relate directly:
Cycle Number to Fluorescence to DNA Starting Concentration
Increased sample accuracy
Reduced errors (monitor reactions more closely)
Faster to optimise/set up/run/analyse

55. Cost Effectiveness of the Rotor-Gene
Usage of 0.2 ml or 0.1 ml tubes
Reduced running costs, no specialized consumables
Free choice of purchasing primers and probes
Reagent contracts are not required
Free Software upgrades

56. Two Categories of Detection Formats
(i) Sequence-unspecific detection using intercalating fluorescent dyes
 SYBR® Green
(ii) Sequence-specific probes

57. Transfer of light energy from an excited (donor-)fluorophor to
FRET I
FRET: Fluorescence-Resonance-Energy-Transfer
Transfer of light energy
from an excited (donor-)fluorophor to
another (acceptor-)fluorphor/non-fluorescent quencher