Eppendorf Rotor-Gene Software manual

Rotor-Gene

Software Manual

Rotor-Gene I

Table of Contents

Foreword 0

Part I

Introduction

................................................................................................................................... 51 Important: Read Before Running The Rotor-Gene

......................................................................................................................................................... 5Rotor-Gene Keyboard (Rotor-Gene 2000 Only)

......................................................................................................................................................... 5Rotor-Gene Startup

......................................................................................................................................................... 6Software Version

......................................................................................................................................................... 7Welcome Screen

Part II Rotor-Gene Description

................................................................................................................................... 91 32-Well and Dual-Channel Rotor-Gene

................................................................................................................................... 102 Multi-Channel Rotor-Gene

................................................................................................................................... 113 Locking Ring (For 36 Well Rotor Only)

Part III

Two Different Rotor Systems (Tubes)

................................................................................................................................... 121 Rotor 36 Well System

................................................................................................................................... 122 Rotor 72 Well System

Part IV Installation and Maintenance

................................................................................................................................... 121 Installation (Rotor-Gene 2000)

................................................................................................................................... 132 Installation (Rotor-Gene 3000)

................................................................................................................................... 133 Maintenance

Part V

Functional Overview

................................................................................................................................... 131 Workspace

................................................................................................................................... 132 Toolbar Workspace

................................................................................................................................... 143 View Raw Channels

................................................................................................................................... 144 Toggle Samples

................................................................................................................................... 155 File Menu

......................................................................................................................................................... 15New Experiment

......................................................................................................................................................... 16Opening and Saving

......................................................................................................................................................... 16Export

......................................................................................................................................................... 17Setup

................................................................................................................................... 186 Analysis Menu

......................................................................................................................................................... 18Analysis Toolbar

......................................................................................................................................................... 18Quantitation

.................................................................................................................................................. 19Reports

.................................................................................................................................................. 19Standard Curve

.................................................................................................................................................. 20Standard Curve Calculation

.................................................................................................................................................. 21Import Standard Curve

.................................................................................................................................................. 21Invert Raw Data

.................................................................................................................................................. 22Calculation of CT

.................................................................................................................................................. 23Results

........................................................................................................................................... 24Dynamic Tube Normalisation

........................................................................................................................................... 24Noise Slope Correction

........................................................................................................................................... 25Ignore First

........................................................................................................................................... 25Quant. Settings

........................................................................................................................................... 25Slope, Amplification, Reaction Efficiency

........................................................................................................................................... 26Offset

........................................................................................................................................... 27Why Are Rotor-Gene Standard Curves Different?

.................................................................................................................................................. 29Main Window

......................................................................................................................................................... 30Melt Curve Analysis

.................................................................................................................................................. 30Sidebar

.................................................................................................................................................. 31Reports

.................................................................................................................................................. 31Results

.................................................................................................................................................. 31Genotyping

......................................................................................................................................................... 32Allelic Discrimination

.................................................................................................................................................. 32Reports

.................................................................................................................................................. 32Results

.................................................................................................................................................. 32Normalisation Options

.................................................................................................................................................. 33Discrimination Threshold

.................................................................................................................................................. 33Genotypes

......................................................................................................................................................... 33Scatter Analysis

.................................................................................................................................................. 34Reports

.................................................................................................................................................. 34Results

.................................................................................................................................................. 34Normalisation Options

.................................................................................................................................................. 35Genotypes

.................................................................................................................................................. 35Scatter Graph

......................................................................................................................................................... 35Comparitive Quantitation

......................................................................................................................................................... 37EndPoint Analysis

.................................................................................................................................................. 38Terms Used In EndPoint Analysis

.................................................................................................................................................. 39Profile Configuration

.................................................................................................................................................. 40Analysis

.................................................................................................................................................. 40Define Controls

.................................................................................................................................................. 41Normalisation

.................................................................................................................................................. 43Threshold

.................................................................................................................................................. 44Genotypes

................................................................................................................................... 447 Run Menu

......................................................................................................................................................... 44Start Run

......................................................................................................................................................... 44Pause Run

......................................................................................................................................................... 44Stop Run

................................................................................................................................... 458 View Menu

......................................................................................................................................................... 45Experiment Settings

.................................................................................................................................................. 45General

.................................................................................................................................................. 46Machine Options

.................................................................................................................................................. 46Messages

.................................................................................................................................................. 47Channels

.................................................................................................................................................. 48Tube Layout

.................................................................................................................................................. 49Security

......................................................................................................................................................... 50Temperature Graph

......................................................................................................................................................... 50Profile Editor

.................................................................................................................................................. 51Hold

.................................................................................................................................................. 51Optical Denature Cycling

IIContents

Rotor-Gene III

........................................................................................................................................... 51What is Optical Denature Cycling?

........................................................................................................................................... 53Configuration

........................................................................................................................................... 53Adding a New Optical Denature Cycling

........................................................................................................................................... 55Changing An Existing Step to Use Optical Denature

.................................................................................................................................................. 56Cycling

.................................................................................................................................................. 56Acquisition

.................................................................................................................................................. 57Melt

......................................................................................................................................................... 57Profile Progress

......................................................................................................................................................... 58Edit Samples

......................................................................................................................................................... 60Gain Calibration

.................................................................................................................................................. 60Auto-Calibration

.................................................................................................................................................. 62Manual Calibration

......................................................................................................................................................... 63Display Options

................................................................................................................................... 649 Gain Menu

................................................................................................................................... 6410 Window Menu

................................................................................................................................... 6411 Help Menu

......................................................................................................................................................... 65Send Support E-Mail

Part VI

General Functions Used Over Several Windows

................................................................................................................................... 651 Opening A Second Experiment

................................................................................................................................... 662 Spanner Icon

................................................................................................................................... 673 Scaling Options

................................................................................................................................... 674 Abbreviations

................................................................................................................................... 675 Auto-Scale

Part VII Quick Start Using The New

Experiment Wizard

................................................................................................................................... 681 Welcome Screen

................................................................................................................................... 692 Page 1

................................................................................................................................... 703 Page 2

................................................................................................................................... 714 Page 3

................................................................................................................................... 725 Page 4

Part VIII

Rotor-Gene Hardware Information

................................................................................................................................... 721 Filter Specifications

......................................................................................................................................................... 7232 Well and Dual-Channel Machines

......................................................................................................................................................... 73Multi-Channel Machines

Part IX Troubleshooting

................................................................................................................................... 741 Log Archives

................................................................................................................................... 742 Troubleshooting Experiment Files

......................................................................................................................................................... 75Initial Denature Step

......................................................................................................................................................... 75Cycling Profile

......................................................................................................................................................... 75Melt Curve Analysis

......................................................................................................................................................... 76Gain Settings

......................................................................................................................................................... 76General Setup Conditions

......................................................................................................................................................... 76Experiment Settings

................................................................................................................................... 773 Regional Settings In Windows 98

IVContents

Part X

Rotor-Gene 3000 Setup Instructions

Part XI Rotor-Gene 2000 Setup

Instructions

Part XII

Rotor-Gene 2000 IMPORTANT SETTINGS

Index

5 Rotor-Gene

Before running the Rotor-Gene you should pay attention to the following:

1) Always run the unit with 36 (32) or 72 tubes in the rotor. If you do not have this many samples load

empty tubes into the rotor. This ensures the thermal load in the chamber is identical for every run. If

0.1ml tubes are used it is sufficient to place empty tubes without lids into the rotor.

Do not use caps on the blank tubes. If caps are loaded on blank tubes and run multiple times

there is a risk that caps may come off the tube causing damage to the rotor.

72-well Locking Ring:

To ensure that tubes sit firmly in the 72 well rotor, a 72 well locking ring is

available. After loading the tubes, the rotor can simply be put over the rotor.

2) Ensure the software is not operating in the Virtual Machine Mode. Go to the

File menu

and select

Setup...

Check that the Virtual Machine box is not ticked.

Setup...

cannot be selected then it has been disabled and your distributor should have preset the

software.

3) Before running a program ensure the Rotor-Gene shows

READY

on the display. If

READY

is not

shown the machine will not set the Gains for the channels and no data will be collected.

The keyboard can be used to set the unit to a temperature and hold. In addition, the Rotor-Gene

keyboard display shows the lid status, either Open or Closed.

Pressing the

Next key

dvances the window forward to the next display and shows Lid Status then

Set Temperature.

Use the

and

Down

keys to change the set temperature, once set press

Start

The system then will go through a Start sequence that turns on the rotor, then the chamber fan and

then the heater.

The unit will heat or cool and hold the set temperature. This is useful if incubation at a particular

temperature is required before thermal cycling samples.

The keyboard can be used to set a manual temperature but is not used in regular operation.

The Rotor-Gene is setup using the following procedure:

1. Turn the desktop PC on and wait for Windows to boot up.

2. Turn on the Rotor-Gene on the left-hand side of the unit wait for the ready message to be displayed

on the LCD.

1 Introduction

1.1 Important: Read Before Running The Rotor-Gene

1.1.1 Rotor-Gene Keyboard (Rotor-Gene 2000 Only)

1.1.2 Rotor-Gene Startup

3. Double click on the Rotor-Gene icon.

4. The Rotor-Gene is now ready for use.

Note for Rotor-Gene 2000 users: If the Rotor-Gene is turned ON without having Rotor-Gene

software loaded and running on the PC, the instrument will emit a buzzing sound. By running

the Rotor-Gene software the buzzing will stop.

1.1.3 Software Version

Software development for the Rotor-Gene system is ongoing. To check on your version number click

Help

then

About Rotor-Gene...

The latest software version is available for download at our web

site.

This screen displays general information about the software, and specifically includes the version of

the software, serial number of the machine, as well as the date it was last updated.

6Introduction

7 Rotor-Gene

This screen appears when a new version of the Rotor-Gene software has been installed and the

Rotor-Gene icon is double clicked for the first time after the installation.

Machine Serial Number:

Type in the serial number (six digits), which is located at the back of the

Rotor-Gene.

Offset Coefficient:

Type in the Offset coefficient, which can be found next to the serial number of the

machine. If no number can be found, the value 0 should be entered.

Machine Type:

Chose your type of machine.

1.1.4 Welcome Screen

8Introduction

Choose type of 36 well Rotor:

Run in Virtual Mode

(for demonstration): Ticking the box allows installing the Rotor-Gene software

on a computer without Rotor-Gene attached. The software is fully functional and can even simulate

runs.

NOTE: If this box is ticked with a Rotor-Gene connected to your computer, a message will

appear before you start your run: "you about to run in virtual mode". To be able to perform a

"real" run, the setup (see setup) has to be changed.

Begin:

When all parameters are set, press Begin. A window will come up initializing machine. Wait

until the machine is initialized, which might take a few seconds. If virtual mode was chosen the

following screen appears:

If the box stays unticked the machine is initialized and opens up the Rotor-Gene software

automatically.

What's new:

This topic explains the new features in this version of Rotor-Gene and answers

commonly asked questions about the location of existing features that have been moved. These

features incorporate design changes and requests by users.

Exit Program:

Exits program.

9 Rotor-Gene

The Rotor-Gene is a real-time thermal cycling system used for DNA amplification and hybridization.

The unit holds 36 x 0.2ml standard micro-fuge tubes (32 x 0.2), which are loaded into a 36-position

rotor (32). With a Dual-Channel machine, a 72-well rotor is also supplied that can run special strip

tubes that are 0.1ml in strips of four. During the run the rotor spins at approximately 500 rpm as the

tubes are thermally cycled in a low-mass air oven.

NOTE: Always run a full rotor of tubes, for both the 72-well and 36-well rotors. If the number

of samples is small fill the remaining positions with empty tubes. This ensures the thermal

load of plastic tubes is the same from run to run.

There are two detection modules in the Rotor-Gene, Channel (1) is setup to detect 510nm and

Channel (2) is setup to detect 555nm. This allows for multiplexed samples to be run.

As the sample tubes spin past the detector modules, a high powered LED strobes the sample and a

photomultiplier (PMP) collects the fluorescent energy. See the figure below.

This data is sent to a Desktop PC that averages the energy of each sample over a number of

revolutions. This data is then displayed in real-time on the screen as fluorescence versus cycle

number or temperature plot.

2 Rotor-Gene Description

2.1 32-Well and Dual-Channel Rotor-Gene

2.2 Multi-Channel Rotor-Gene

The principal of the Multi-Channel Rotor-Gene is very similar to the 32-well and the Dual-Channel

Rotor-Gene. However, the Multi-Channel has one LED with four excitation filters.

From the side view of the heating cooling chamber we see the LED source irradiating the tube from the

side wall and the photomultiplier detecting the energy from the base of the chamber. The detection

filter wheel has six different filters, four are band-pass that are used in four Channel multiplex runs, the

other two are high pass filters used with other non-standard fluorophores.

10Rotor-Gene Description

11 Rotor-Gene

Opening and closing 0.2 tubes several times before running might loosen the closing mechanism of

tubes. The 0.2 tubes run on the 36-well rotor on the Rotor-Gene should only be closed once before

running, otherwise it might result in popping of tubes.

Also make sure that the 0.2ml tubes are

closed properly by firmly pressing down the lid of the tube.

If popping is experienced or wants to

be avoided, a locking ring can be used on the Rotor-Gene.

The Locking Ring is designed to use

for flat top 0.2 ml tubes only.

The below graph shows schematically how the locking ring has to be used:

1) Shows the schematic representation of the 36-well rotor.

2) Screw down the screw with threads on both sides. Make sure the nipple and the red dot faces the

top.

3) Simply place the locking ring over the top of the tubes.

4) Screw down the locking ring holder (4) over the screw with threads on both ends (2) until the

locking ring (3) is fixed.

2.3 Locking Ring (For 36 Well Rotor Only)

3 Two Different Rotor Systems (Tubes)

It is recommend that you use flat cap, 0.2ml tubes from Axygen

, supplied by Corbett Research.

If using your own tubes, be aware of the following:

1. The tube cap must fit down tightly otherwise there is a risk that the cap may 'pop' open during

thermal cycling. This has been observed with some domed capped 0.2ml tubes. If necessary the

locking ring could be used to secure the tubes.

2. Be sure that the tubes fit into the rotor firmly. Tubes that are excessively loose may vibrate during a

run and interfere with the data acquisition and give poor results.

3. In addition, ensure that the tubes are not too large. If the diameter of the tube is too large it will not fit

down far enough into the rotor. Samples will not be optically aligned over the detection system and may

cause a reduction in fluorescent signal acquired and sensitivity.

4. Do not use tubes from different manufacturers within the same run. This may result in

inconsistencies.

5. Locking Ring can only be used with

flat top

tubes.

Corbett Research manufactures the 72-well tubes and caps in strips of four especially for the Rotor-

Gene.

It is not advised to autoclave the 72-well tubes and strip caps as they may distort.

The tubes can become visibly deformed and are no longer straight but bent slightly. When running

tubes that have been autoclaved it has been noted that the caps may detach from the tube.

This has the effect of the loose caps being circulated around the chamber causing damage to the

insulation material. It is also advisable to exchange the empty 72-well tubes after at least ten runs.

Caps might start popping.

Do not use caps on the blank tubes. If caps are loaded on blank tubes and run multiple times

there is a risk that caps may come off the tube causing damage to the rotor.

The Rotor-Gene 2000 and the Desktop PC should be connected with the special "Y" mains cable

supplied. This special cable minimizes the connection length between the two devices.

NOTE FOR Rotor-Gene 2000 Users: If you do not set up the mains connection as described

you may see spikes in the collected data due to mains noise.

3.1 Rotor 36 Well System

12Two Different Rotor Systems (Tubes)

3.2 Rotor 72 Well System

4 Installation and Maintenance

4.1 Installation (Rotor-Gene 2000)

13 Rotor-Gene

No special installation steps are required for the Rotor-Gene 3000. The Rotor-Gene 3000 requires

only a standard serial cable to be connected to a communications port on the back of the Laptop or

Desktop computer.

The only maintenance the user may have to perform is keeping the lenses relatively clean and free

from dust that may build up over time.

It is recommended that when the Rotor-Gene is not in use, the lid is in the closed position to minimize

dust falling into the heater chamber.

Looking down into the heating chamber you will notice one (36/72-Well, Multi-Channel) or two (36/72-

Well, Dual-Channel) round lenses, which are the detector objectives for the different Channels. Should

those lenses be covered with dust or any other debris you should clean them using a cotton bud and

ethyl alcohol.

On the side-wall of the heater chamber there are also one (36/72-Well, Multi-Channel) or two (36/72-

Well, Dual-Channel) round lenses for the high power diode emitter. These lenses are not likely to build

up dust, however, if you place tubes into the rotor that are dirty or wet you are likely to spin those

particles around the inner wall of the heater chamber and possibly produce a build up on these lenses.

If these lenses need to be cleaned, the rotor can be removed by unscrewing the black lock-down in the

center and removing the rotor. These lenses can be cleaned with a cotton bud and ethyl alcohol.

The following chapter will help to familiarize you with elements in the Rotor-Gene user interface.

The Rotor-Gene workspace is the backdrop of the main window. This is the area in which you can

open up graphs of raw data, temperature and analysis results. If you have several windows currently

open, you can organize them by clicking the

Arrange button

on the toolbar. There are several

additional options available, which you can access by clicking on the

Down Arrow

, which appears

next to the button.

These buttons are shortcuts to frequently used operations. To see what their function is, hover your

mouse over them and a brief explanation will appear. These commands can also be accessed via their

corresponding menu items with the same name.

4.2 Installation (Rotor-Gene 3000)

4.3 Maintenance

5 Functional Overview

5.1 Workspace

5.2 Toolbar Workspace

5.3 View Raw Channels

Click on these buttons to view different channels in the experiment.

Use this control to hide or show different samples. Semi-greyed samples are deselected. The

Scroll

Bar

is used to display the next group of samples. You can toggle samples individually by clicking on

them, or you can hide/show all currently visible (on the current 'bank') by clicking on

Bank on/ Bank

off

. To select a range of samples, click on the first sample and drag your mouse to the end 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. Clicking

All On /All Off

will show all/ none 36 or 72 samples. Pressing the

Edit Samples…

button opens the samples window.

Toggle samples ID display:

If a 72-well rotor is used the samples are shown from A1 to A8, B1 to

B8, etc. Using the toggle samples ID display button lets the user switch to a numerical order of

samples (1 to 72).

The sample toggler:

5.4 Toggle Samples

14Functional Overview

15 Rotor-Gene

This screen presents you with a selection of available experiment templates. In a laboratory

environment, you can set up standard templates by placing them in the Rotor-Gene template directory

(usually C:\Program Files\Rotor-Gene\Templates). These templates will then appear in this window.

New Experiment:

Double-clicking this icon will create a new experiment based on the last experiment

run. To always begin with a blank experiment, choose File, Save As and save a template in the Rotor-

Gene template directory as "Blank Template.ret". Then, double-click this icon instead of New

Experiment when starting a new run.

New...:

Opens the template selection window to create a new experiment.

Browse...:

Pressing Browse... opens a window in which you can select the template you would like to

use for the next run. This option is also available under Other Templates.

Other Templates...:

This icon has the same effect as clicking the Browse... button, and displays an

open dialogue to load custom experiment templates.

Cancel:

Closes this window.

Help:

Opens on-line help.

Use Experiment Wizard to Configure Experiment:

Upon selecting a template, the New Experiment

Wizard will be displayed. Unticking the Use Experiment Wizard option can disable the wizard.

Show this screen when Rotor-Gene opens:

Untick the box if you don't want to see this window

coming up before using the Wizard.

5.5 File Menu

5.5.1 New Experiment

5.5.2 Opening and Saving

Open...:

Opens a previously saved Rotor-Gene Experiment (REX file) or Rotor-Gene Experiment

Archive (REA file).

Open Recent...:

Displays the last four files you have opened or saved.

Save:

Saves the experiment, prompting you for a filename if this is the first time the experiment has

been run. You have the option of saving the experiment as a template, meaning that you can base

future experiments on it. You can also save in the Rotor-Gene Experiment Archive (REA) format that

compresses the experiment. This is ideal for emailing, as it does not need to be compressed before

sending.

Save As...:

Saves the experiment, always prompting you for a filename. It also lets you choose what

type of file you want to save the document as.

A file can be saved as an experiment, a template, an experiment archive or an experiment in a previous

RG software version (4.3).

Opens the Export window where you can export channel and sample data to a format readable by

other software. You can export Rotor-Gene data to third party software such as spreadsheet and

statistical applications. This screen allows you to export channel information and the sample list.

Export Samples:

The experiment's sample list will be saved in a format, which can be opened by

popular spreadsheet applications. Once you have saved the samples, drag the export file into your

spreadsheet.

Export Channels:

You can export both raw data obtained from the machine and the data used to

perform analysis after normalization and other operations have been performed. Select the channel to

export, and the type of data you wish to export, then click Export. The resultant file can be opened by

dragging it into the target application.

16Functional Overview

5.5.3 Export

17 Rotor-Gene

Choosing File, Setup... opens the Rotor-Gene setup window.

Machine Type:

Select the machine you are operating from the option buttons available.

Virtual Machine:

This option enables you to operate the software even when a machine is not

attached for demonstration purposes.

Allow access to this screen:

Untick this box, once the machine has been correctly selected to

prevent users from altering this setting. If you need to have access to this screen again, contact your

distributor.

5.5.4 Setup

5.6 Analysis Menu

This toolbar enables you to create new analyses and displays existing ones. The tabs at the top of the

bar enable you to select a method of analysis to perform. Once you have done this, a list of the

channels which can be analyzed using this method will be listed below. Those that have already been

analyzed will have a green tick next to them, such as the first two channels shown in the screen shot.

This means that threshold and normalization settings have been remembered for this analysis. To

analyze a channel, or view an existing channel, simply double-click on the channel to view. The

specific analysis window will then appear.

Auto-shrink Window:

Ticking the box shrinks the window when it is not used. Moving the cursor over

the window enlarges the window again.

Organizing Your Workspace:

Each time you double-click on a new analysis, its windows will be

arranged to fit in with those already on the screen. With many windows, this can be difficult to use.

Simply close the windows you do not require, then click

Arrange

on the toolbar. The windows will

automatically be rearranged according to the

Smart Tiling

method. You can select another method of

arrangement by clicking the

down arrow

next to the toolbar button.

Clicking the right mouse button over the analysis window also allows to

Show, Hide

Remove

Analysis

Double click quantitation or press

Show

to open the channel of interest. Three windows will be opened

automatically, the main screen, the standard curve and the results. Going from left to right, the

following buttons are displayed in the main window:

5.6.1 Analysis Toolbar

18Functional Overview

5.6.2 Quantitation

19 Rotor-Gene

Reports:

Opens the Quantitation Report selection window where you can choose a report to preview

of the currently selected Quantitation analysis. There are three different options, Standard Report, Full

Report and Concise Report.

Using the buttons on the top, the reports can be

printed, saved, emailed

exported to Word

Std. Curve:

This button opens the standard curve graph. By default, this window is opened when an

analysis is opened. If you close the window, it can be re-opened by using this command.

On the standard curve the values are recalculated dynamically as the threshold level is varied, by

clicking and dragging.

Blue dots

on the curve show the samples that have been defined as standards and

red dots

show

the unknown sample data points.

NOTE: If redefining standards to recalculate the standard curve, toggling the standard colors

ON or OFF will remove it from standard curve calculation.

Efficiency:

The Reaction Efficiency of the experiment. This value is discussed in more detail in

Slope,

Amplification, Reaction Efficiency

R^2-value

(correlation coefficient):

The R^2 value, or R

value (as displayed with the superscript),

is the percentage of the data which is consistent with the statistical hypothesis. In the Quantitation

context, this is the percentage of the data which matches the hypothesis that the given standards form

a standard curve. If the R

value is low, then the given standards cannot be easily fit onto a line of best

fit. This means that the results obtained (ie. the calculated concentrations) may not be reliable. A good

R2-value is around 0.99.

NB: It is still possible to achieve a high R^2 value with a poor standard curve, if not enough

Reports

Standard Curve

20Functional Overview

standards have been run. The R^2 value will improve as the number of standards decreases.

R-value

(square root of correlation coefficient):

The R-value of the calculation is the square root

of the R^2 value. Unless you have a specific statistical application, the R2 value is more useful in

determining correlation.

According to the formula

y = mx + b

the slope (

) and the intercept (

) of a standard curve are

automatically calculated and shown in the top right corner of the standard curve window.

Export to JEPG...:

With the pointer on the standard curve, click on the right button to show the option

to export as a JEPG.

Singlet Concentration:

This function is used when no replicates on a standard have been run or

defined. With the function enabled the standard curve shows a line between the Given concentration

and the Calculated concentration for the standard series. The threshold is then optimized, by moving

the level to minimize the distance between each Given and Calculated standard concentration.

Overlay:

When multiple quantitation runs have been performed in the same experiment, it is possible

to overlay the standard curves in the same window. This is useful for graphically viewing the difference

between different thresholds on the statistical results. Below is a screenshot of this feature:

"conc=...*CT + ..."

represents the equation used to relate CT values and concentrations. Type can be

either floating or fixed. If floating, an optimal standard curve equation is calculated as you move the

threshold. If Fixed, the equation does not change because it has been imported from another

experiment.

Note: The Rotor-Gene standard curve formula is displayed in a different format to

competitors' standard curves. Competitors express their curve in terms of concentrations

instead of CT values, which does not affect results, but does affect the display of the M

(gradient) and B (offset) values. Please read the section

Why Are Rotor-Gene Standard

Curves Different?

for more information.

Standard Curve Calculation

21 Rotor-Gene

Importing a standard curve allows you to perform estimates of concentrations when a standard curve

is not available in an experiment and you are certain that the reaction efficiency has not varied between

the two runs. You can import curves from another channel, or from another experiment by clicking on

Import Curve.

You can choose to

Adjust

the standard curve, or not to adjust. Adjusting means modifying the offset

(b) in the standard curve to be consistent with the current experiment. Whether you should adjust the

standard curve or not depends on the chemistry application.

A reference should be defined in the target experiment to adjust the curve to match the target

experiment. You can define a reference by setting a sample's type to Standard, and entering a

concentration value in the Sample Editor. Multiple copies of the same reference can be entered to

improve accuracy of the method. Note that you cannot define more than 1 reference concentration. For

example, it is possible to have 3 replicate references of 1000 copies, but not to have one reference of

1000 copies, and another with 100 copies in the same experiment.

Once the curve has been imported, the standard curve type will be changed to Fixed. Click

Reset

set the curve type back to Floating.

Below is a screenshot of the Import Standard Curve screen:

Using this screen, you can import the standard curve from another channel you have analyzed in the

current experiment, or you can load a standard curve from another experiment.

Current Experiment:

When this option button is selected, quantitation analyses on other channels

will be listed with their corresponding standard curves.

From Other Experiment:

Selecting this option button will bring up an Open Dialog in which you can

select an experiment to open. If any quantitation analysis has been performed for the experiment, you

will see standard curves listed for each channel analyzed.

Channels List:

Lists the analyzed channels and their respective standard curve formulas.

Some chemistries produce a fluorescent readout that decreases exponentially instead of increasing. It

is still possible to analyse these using Quantitation, but the "Invert Raw Data" checkbox should be

ticked.

For all other quantitation analysis, this option must remain unticked.

Import Standard Curve

Invert Raw Data

NB: Techniques such as Quenched FRET

or the use of Bodipy

which use decreasing

signals to quantitate may have less accurate results than those that increase. Such

techniques have not been widely verified yet in the scientific community.

Calculation of CT

Ct-Calculation:

The Ct value is the value where the threshold line crosses the amplification curve. By

setting a threshold line and calculating the intersection with each of the sample curves, the Ct values

for each sample are established.

Threshold:

(Manual Set): To set the threshold click on the icon (grid with red arrow) then click and

hold on the graph and drag a threshold line to the desired level, or enter a log value. Alternatively the

Auto-Find Threshold function can be used to automatically determine the best level. When setting a

threshold manually, it should be set in the exponential phase of the experiment, significantly above the

background level to avoid noise.

Auto Find Threshold:

The automatic threshold function will scan the darkened region of the graph to

find a threshold setting which delivers optimal estimates of given concentrations. You can change the

region to be modified by entering new upper and lower bounds in the text boxes. For most quantitation

analyses, the default region is suitable. Based on the standards that have been defined the function

then scans the range of threshold levels to obtain the best fit of the standard curve through the

samples that have been defined as standards, (i.e. maximizes the R value to approach 1.0).

Eliminate Cycles before:

To set click on the icon (grid with red arrow) then click and hold on the

graph and drag the threshold line to the right. This eliminates the threshold line for low cycle numbers.

Note: This is useful when there is noise on the signal during the initial cycles.

22Functional Overview

23 Rotor-Gene

Opens the quantitation results grid. By default, this window is opened when you open an analysis. If

you close it, it can be re-opened by using this command.

The results obtained from the experiment are summarized in a table. Clicking the right mouse button,

and selecting

Export to Excel

will export the table to Excel. There is no need to open the Excel

program, as this will be done automatically.If you would like to copy the data into an existing

spreadsheet, choose the

Copy

option instead, then open your spreadsheet, then select paste.

To make calculations easier, a feature called

AutoStat

is introduced which automatically calculates

the Average, Standard deviation, Minimum and Maximum values of samples of interest. Simply move

the mouse over the area of interest and the values are given in a small table displayed below the

sample list on the right-hand side of the screen.

Results

24Functional Overview

In this screenshot, samples 1 to 5 are analysed:

This option is ticked by default and is used to determine the average background of each individual

sample just before amplification commences. Standard Normalization simply takes the first five cycles

and uses this as an indicator for the 'background' level of each sample. All data points for the sample

are then divided by this value to normalize the data. This process is then repeated for all samples. This

can be inaccurate as for some samples the background level over the first five cycles may not be

indicative of the background level just prior to amplification. Dynamic Tube Normalization uses the

second derivative of each sample trace to determine a starting point for each sample. The background

level is then averaged from cycle 1 up to this starting cycle number for each sample.

This method

gives the most precise quantitation results.

Alternatively with some data sets it may be necessary

to disable the dynamic tube normalization. If this is the case the average background for each of the

samples is only calculated over the first 5 cycles. If the background is not constant over the cycles

before amplification it will result in less precise data.

The background fluorescence (Fl) of a sample should ideally remain constant before amplification.

However, sometimes the Fl-level can show an increase or decrease due to the effect of the chemistry

being run and produce a skewed noise level. The Noise Slope Correction option uses a line-of-best-fit

to determine the noise level instead of an average, and normalizes to that instead. Turning on this

option can tighten replicates if your sample baselines are noticeably sloped.

This function improves the data when raw data backgrounds are seen to slope upward or downward

before the amplification take off point (Ct). It is very helpful for experiments when for example the FAM

background is seen to creep upwards over the initial cycles.

Dynamic Tube Normalisation

Noise Slope Correction

+ 56 hidden pages

Eppendorf Rotor-Gene Software manual

Specifications and Main Features

Frequently Asked Questions

User Manual