Rotor gene manual

Quantification of Gene Expression This analysis is based on assessment of Ct values obtained through manual threshold setting by the user. Each fluorescent channel needs to be evaluated individually. Scatter Graph Analysis This type of analysis enables quick and simple evaluation of obtained values through a chart.

Used Corbett rotor-gene RG , was working when taken out of service. Data security, from starting the run to exporting the results, is assured by audit trails, digital signatures, and individual user management.

Each Rotor-Gene Q MDx instrument is supplied with an unlimited software user license for your institute, enabling you to access and analyze your test results on as many computers as you want — in the laboratory, or in the office.

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View Quote Example. Catalog Number. Looking for a quick way to design experiments? To position the Gene-Disc into the loading block, use the position one tab on the Gene-Disc. Slide the Gene-Disc into the loading block by using the position one tab and tube guide holes on the loading block. The design of the loading block allows for easy hand pipetting. Leaving the Gene-Disc in the loading block, obtain one sheet of heat sealing film and remove the central portion.

Do this by slightly folding the film in half, pinching the centre piece and carefully tearing it out. The central hole of the film should slide comfortably over the cylinder of the loading block and onto the top of the Gene-Disc. Turn on the heat sealer using the rocker switch located at the back left hand side. A red Power light will illuminate on the top of the heat sealer.

The heat sealer should take about ten minutes to reach operating temperature, following which a green Ready light will illuminate. Once the heat sealer is ready it is safe to leave it running constantly.

Slide in the loading block Gene-Disc film assembly using the guide rails on the side of the loading block. Ensure that the loading block is pushed in all the way. Do this by pressing down on the blue anodised bar at the top front of the unit with your palm and pushing the black catch. When the mechanism has lowered, an orange Sealing light will illuminate on the top of the unit. If the Gene-Disc block is not in the correct position a warning beep will be heard.

When ready, the unit will beep and the orange ready light will illuminate, press down on the blue anodised bar to raise and lock the heater mechanism back to the ready position. Do not leave it any longer than indicated by the beep or the Gene-Disc may deform.

Slide the loading block out of the heat sealer. Allow the film to cool for approximately 10 seconds, and then tear off the excess film. Remove the Gene-Disc from the loading block. To load the Gene-Disc into the rotor, use the position one locator tab to find the correct orientation. The quick start wizard is designed to allow the user to start the run as fast as possible, the advanced wizard has more options available such as configuring gain optimisation settings.

For convenience the wizards contain a number of templates which have default cycling conditions and acquisition channels. To change the type of wizard used you can change the tab located at the top of the New Run window. The user can select from a set on commonly used templates entering the bare minimum of parameters to get started.

The first step is to select the desired template for the run. Perform Last Run imports the cycling and acquisition and sample definitions from the last run open in the software.

Three Step with Melt: three step cycling profile and a melt curve with data acquisition on the Green channel. Nucleic Acid Concentration Measurement is a default template for measuring the concentration of nucleic acid using intercalating dyes. The cycling and acquisition profiles can be altered during the wizard for all templates. TIP User defined templates can also be added to the initial quick start template list by copying.

After copying them to this path the template will appear as a icon in the software. To have custom icons for your templates, create a. ICO image with the same file name as the template. It is even possible to create subfolders in the templates folder to group related templates.

This allows you to organise your templates if many users are using the same machine, for example. Tick the Locking Ring Attached option to proceed through the wizard. This can be altered using the Profile Editor. To initiate a run click the Start Run button. The ability to save templates is also available before starting the run.

The run can be saved in the user's desired destination. The run is given a filename with the template used and the date of the run. A serial number is also designated to allow automatic naming of numerous runs that use the same template on the same day.

For information about setting up sample definitions see Sample Editor. Select a template from the Advanced tab of the wizard. Template options provided in the advanced templates window are similar to the quick start selection, the differences between them are listed here. Empty Run: An empty run allowing the user to start defining their profile from scratch. Two Step: two step cycling profile with data acquired on the Green channel only to speed up the run.

HRM: High resolution melt curve profile. Other Runs: Conventional melt and hybridisation profile. This template is locked to ensure the profile will always operate correctly. Similar to the Quick Start Templates copying. As in the quick start wizard, you must acknowledge the displayed warning before continuing. The reaction volume must be entered. The Rotor-Gene is configured for mL volumes.

The software recommends longer holds during cycling for larger volumes. If the 72 well Carousel is selected, three Sample Layout formats are available. If consecutive samples are in fact in every 8'th well if loaded with a multi channel pipette the A1, A2, A However, most users will use the default option 1, 2, Click the Edit After setting up the profile click the Gain Optimisation The initial cycling profile is based on the template selected Setting up a Run.

The profile is graphically displayed. The list of the segments that the profile consists of is listed below the graphical display. If the instrument contains a HRM channel. The settings of each cycle of the profile can be edited by clicking on the cycle in the graphical display or the name of the cycle in the list and changing the set temperatures and hold times.

Insert after Insert before Remove: removes the selected cycle from the profile. Cycling Long Range option adds 1 second to the hold time at each cycle for a user defined number of cycles. Melt and Hybridisation A Melt step is a ramp between two temperatures, from a lower to a higher temperature.

If the temperature is set to decrease, the step is labelled as Hybridisation instead. To set up a melt, define the start temperature, the end temperature, the length of time to hold at the first acquisition temperature before the ramp is initiated, the temperature increments, the time each increment is to be held for, and the acquisition channels.

The high resolution melt step will always acquire data using the HRM sources and detectors, it also has the option to perform gain optimisation just before the melt begins. After performing a high resolution melt the data can then be analysed with the HRM Analysis module.

To change the temperature click the Hold Temperature button and type or use the slide bar to change the temperature. Similarly to change the duration of the hold click on the Hold Time, mins and secs buttons. If running an Optical Denaturation, you can elect a hold step as a calibration step. This causes a calibration melt to be performed before this step. By default, this is configured for the first hold in the run, but may be overridden if you are using 60 degree preholds before your 95 degree hold.

The number of repeats is set using the This cycle repeats X time s selector. The individual repeat is displayed as seen in bottom right of the graphic below.

Each step of the repeat can be altered. Temperature can be changed by dragging the temperature bar up or down. The duration of the step can be changed by dragging the temperature boundary left or right. Alternatively, click on the step and use the temperature and time buttons to the left of the repeat display. Touchdown: Touchdown can be enabled to decrease the temperature during the initial cycles. Set the number of cycles and the program decreases the temperature by the specified number of degrees every cycle.

This is reflected in the graphical cycle representation. Click on the Not Acquiring button arrowed above. If a channel has been already been set to acquire at this step, then the acquiring channels will be listed here instead. Once clicked, the Acquisition window will appear. To set a channel to acquire, move the channel from the Available Channels list to the Acquiring Channels list using the button.

To remove a channel from the acquiring channels list, use the button. The button removes all the channels from the acquiring channels list. Clicking the Don't Acquire button also removes all acquisitions from the step. If more than one Cycling sequence is included in the profile, the acquired data can be appended to the data acquired from the earlier Cycling.

Use the drop down menu in the Same as Previous option to select the cycling step to which the data is to be appended. Dye Channel Selection Chart: A chart is provided that will help the user decide which channel they should be using. The user can look up the dye they are intending to use, and find the appropriate channel.

The dyes shown in the table are ones that are commonly used, and by no means indicate the limits of the instrument. Melt and Hybridisation For a melt cycle, specify a start temperature, an end temperature, the time you want to wait at before the first acquiring point, the amount of time to remain at each point and a ramp will be generated going between the two temperatures.

If the start temperature is higher than the end temperature, the name of the step will change to Hybridisation. The Acquiring To option, here set to Melt A, can be changed by clicking on the button. The same screen as for Cycling will appear and the channels to acquire to can be selected.

The minimum hold time between temperature steps will vary depending on the number of degrees between each step. High Resolution Melts A high resolution melt step is very similar to a Melt step in the way that the temperature profile characteristics are set. The difference is that only the HRM channel can be acquired on. The HRM step also has the option of automatic gain optimisation just before the melt starts.

The gain instrument sensitivity will be optimised such that highest non-saturated fluorescence is used ie. This will provide the best environment for detecting the entire melt transition and with the highest accuracy.

Optical Denature Cycling is an exciting new technique, available only on the Rotor-Gene , which performs real-time melt analysis to determine the melt peak of a reference sample. This indicates with greater precision when your product has denatured than by setting a particular denature temperature for a hold time.

To perform this technique, simply place a tube of preamplified product in tube position 1 in your rotor.

The reference tube must also contain a detection chemistry that enables strand dissociation to be detected. When first heating the machine to the initial denature temperature, a melt on the Green channel is performed starting from 80 degrees until 95 degrees by default.

The parameters of this initial melt can be adjusted by the user. Then, every Optical Denature step, the machine is heated as quickly as possible and data acquired continuously.

Once the Reference Tube has reached this denature threshold fluorescence level, the machine is immediately cooled and will proceed to the next programmed step in the cycle. While cycling, a peak is not calculated, rather, it is the fluorescence level that is referenced to the melt peak and designates the denature threshold. In this graph, the raw fluorescence readings and the first derivative have been overlayed. It shows the correspondence between the Denature Threshold and the Melt Peak obtained during the calibration.

What do I need to perform an Optical Denature run? This sample should contain the same product as samples of interest and a detection chemistry that allows for the monitoring of product dissociation. What will I see when performing an Optical Denature run? An Optical Denature run, from the user perspective, appears almost identical. The most striking differences are the melt step automatically inserted at the beginning of the profile, and the sharp profile of the denature step during cycling.

The denature step does not require defined hold times as the dissociation of the product is monitored at each cycle. This is the same temperature as your denature step in a standard cycling profile. You must also define an Optical Denature cycling profile. This is enabled by default in this version of the software.

However, your existing templates from older versions of the software will not use this step. Then click New. A default profile containing a Denature step and an Optical Denature Cycling step will appear: The profile is displayed as: The blue shaded region at the beginning of the run represents the optical denature calibration process.

The Green dots represent the acquisitions taken each cycle during heating. The lighter blue dots represent the acquisition at the end of the anneal step at 60 degrees. Note that while the profile shows each step going to the same denature temperature, this may not be the case. If the sample requires slightly longer to melt towards the end of the run, the optical denature process will wait for the melt in the fluorescent data, and not off the times.

For this reason, the temperature trace for optical denature may vary for each cycle. By clicking on the Cycling step, the information about the 2-step profile appears in the lower half of the Profile Editor window. Click on the second half of the graph with the Optical Denature symbol. You can, however, modify it by clicking Edit.

This is because the calibration process and denaturation are equivalent in Optical Denature Cycling. Then, select the denature hold by clicking on it in the preview graph at the bottom of the screen: Click on the drop-down menu here displaying "Timed Step" and select "Optical Denature".

The Temperature and Hold Time will be removed and the Optical Denature icon will be displayed in their place. Gain Optimisation When setting up a new run with reactions that have not previously been run on the Rotor-Gene it is helpful to use the Gain Optimisation function.

This screen allows you to set the Gains of each of the channels and the set temperature. This can be due to changes in fluorescence after the first hold step. Nevertheless, the result of the gain optimisation will give you a good indication on what fluorescence level the run will be started. This is then placed in the machine and Gain Optimisation is run to determine the best Gain setting. Auto-Gain Optimisation This window lets you optimise your instrument by automatically adjusting your Gain settings until the readings for all selected channels fall below a certain threshold.

You can select to optimise all channels, or just those that you will be using in the current run. Set temperature to Before reading, the machine will be heated or cooled to match the given temperature. Selecting "Optimise Acquiring" will instead only optimise those that you have used in the thermal profile defined in the run cycling and melt.

Channel Settings: This is a drop-down menu allowing you to add additional channels to the gain optimisation window. Choose the channel of interest and press Add. Opens a window where the fluorescence range of the sample can be determined. The auto-optimisation process begins on Gain 5, reading from each channel. It chooses the first Gain, which has a fluorescence reading equal to or below the level you set in this box. In the example below, tube position 1 was chosen with a target sample range between 5 and 10 Fl fluorescence units.

Remove and Remove all: Removes the highlighted channel or all channels. Start: Begins the optimisation process. A gain will be chosen which will produce fluorescence signal levels within the specified range. If fluorescence fall outside the specified range, the gain will be set to give the closest match possible. Manual: Opens the Manual Gain Adjustment screen see below. Perform Optimisation Before 1st Acquisition: This tick box performs the Gain Optimisation at the first cycle where data acquisition occurs.

This setting is generally the recommended auto-gain option. The machine is heated to the specified temperature, the gain optimisation is performed, and then cycling begins on the first step, usually a Denature. This can be useful when a optimisation during the run may impact too much on the time spent on the initial step.

Usually, however, Perform Optimisation Before 1st Acquisition is preferred as optimisation is performed as close as possible to run conditions. Changing Gain During a Run: If the Gain at the beginning of the run was accidentally chosen to high or to low it can be changed within the first ten cycles. A vertical line in the main screen will appear where the Gain has been changed. Due to the sudden drop or increase in fluorescence, the cycles before the change will be excluded from the analysis.

Manual Gain Adjustment This window lets you view in real-time the fluorescent readings at any given temperature. It is used when the background of a sample is unknown and therefore the gain must be determined to ensure the sample signal is sufficient to detect.

The display samples can be toggled using the right-hand selector as in the main experiment workspace. It takes approximately 4 seconds to acquire data per channel. During this time the user interface is deactivated and so it is best to 1 Start the test 2 wait for the temperature to stabilize 3 note the end point fluorescence Fl reading 4 stop the unit 5 make the appropriate Gain change and 6 restart the unit.

Temperature: Change this value to set the temperature of the machine. Adjust the temperature on the Gain Adjustment screen to reflect the required acquisition temperature for the run. NOTE The temperature will not be adjusted while the machine is operating. You should restart the machine to apply changes made to the temperature.

Edit Gains: Opens the Edit Gains window. Start: Begins the run, setting the machine temperature to that displayed on the screen. The temperature and channel graphs will start to display data. Stop: Stops the run. If the run is still acquiring data when you click the button, then the machine will first finish acquiring, and then stop the machine.

This process can take up to 5 seconds for each acquiring channel. If the Gain was chosen too low the signal will be lost in background noise, too high and all signal will be lost off scale saturated.

Always check the parameters. If you are satisfied click Start Run and you will be prompted for a file name. The functionality of this screen is identical to the Sample Editor. You may also elect to complete sample information after the run has finished. The Finish and Lock Samples button is optional. It allows you to close the screen and prevent the sample names from being modified. For more information about this and other security features see Security Menu.

This is the area in which you can open up raw data plots, temperature and analysis results. If you have several windows opened concurrently, you can organize them by clicking the Arrange button on the toolbar. There are several window arrangement options available that you can access by clicking on the Down Arrow next to that button.

These commands can also be accessed via their corresponding menu items of the same name. When viewing channel data, a number of options are available to change the presentation of the data. The channels may also be transformed to facilitate different types of analysis.

Adjust Scale will bring up a window in which you can manually enter a scale, or interactively select one. To chose this option simply press the right mouse button over the appropriate screen. Auto-Scale attempts to fit the scale to the maximum and minimum readings in the data. Spanner icon: See the section Spanner Icon for more information on the options provided by this button. Options: Displays the drop-down menu indicated above, providing a number of options for transformation of the raw data.

Normalise to Crop start cycles: Creates a new channel data set in which some start cycles have been removed.

This is useful if large jumps are observed in the initial cycles, as can occur when using certain chemistries. Crop end cycles: Creates a new channel data set in which some end cycles have been removed. Page 1: Indicates the currently selected Sample Page used to display the raw data plots. The Sample Editor allows for the creation of multiple sample definitions, allowing data to be viewed with varying line thickness, sample definitions and other display options.

This is of particular use if Relative Quantitation is being performed in a single channel, as the operator can easily toggle the view between the Gene of Interest and Housekeeper samples through the definition of two sample pages. Use this control to configure which samples are included in the display and results.

Samples with a cell background in vivid colour are displayed while semi-greyed samples are not. The Scroll Bar is used to display the next group of samples. You can toggle samples by clicking on them, or drag-selecting several at a time. NOTE The number of displayed samples in a bank is dynamic, and depends on the screen space available. To select a range of samples, click on a sample and drag the mouse to another sample.

When you release the mouse button, the selected samples will either be toggled on or off. Clicking Named On will only show those samples you have given a name to; a quick way to show only relevant samples. Pressing the Edit Samples… button opens the sample editor window where sample names, types and standards concentrations can be edited see Edit Samples.

Page: This label at the top of the selector indicates the sample page that you are viewing. Pages allow varied independent analyses from the one channel data set. For example, you can run two standard curves in the Green channel and generate independent reports. More information on setting up sample pages is available in the Edit Samples section of the manual. Using the toggle samples ID display button lets the user switch to a numerical order of samples 1 to This ensures that only samples relevant for the analysis of the particular page are displayed.

Select Groups: If you have defined sample groups, this feature will toggle the display of these samples for the active page.

Groups are arbitrary collections of samples that allow advanced reporting of statistical results. You can, for example, define groups of Treated and Untreated patient samples.

Groups can be set up in the Edit Samples window. The sample toggler, shown below. Once a selection has been made the wizard will guide you through the run setup, and will allow you to modify settings and profiles. New Run New Cancel: Closes this window.

Help: Opens on-line help. Show this screen when software opens: If this option is ticked the wizard is displayed when the software is initiated. Open Recent Save: Saves any changes that have been made to a run file.

Save As The options are as follows: Run File The user can change the name and the save location of the run. This is the default format. The template can be used to initiate future runs. Run Archive You should save files in this format before they are emailed. This both reduces the time required to send the file, and ensures that files are not corrupted by mail clients. Unique rotary design for outstanding performance The unique centrifugal rotary design of the Rotor-Gene Q makes it the most precise and versatile real-time PCR cycler currently available Figure 1.

Each tube spins in a chamber of moving air, keeping all samples at precisely the same temperature during rapid thermal cycling. Detection is similarly uniform.

When each tube aligns with the detection optics, the sample is illuminated and the fluorescent signal is rapidly collected from a single, short optical pathway. This thermal and optical uniformity results in sensitive, precise, and fast With up to 6 channels spanning UV to infrared wavelengths, the cycler delivers the widest optical range currently available Table 1 , and is highly suited for all multiplex applications. Easy routine verification Laboratories may often want to verify thermal accuracy.

For most cyclers, this requires interaction with a service engineer. With the Rotor-Gene Q, this is not necessary. The kit includes a specialized Rotor-Disc filled with temperature-sensitive liquid crystals and dedicated analysis software. The full procedure takes less than 30 minutes. Reliable support for your peace of mind In the unlikely event of any service issues with your Rotor-Gene