1) The document provides instructions for setting up and running RT2 Profiler PCR Arrays on the QIAGEN Rotor Gene Q instrument. It describes how to create a PCR protocol template, perform real-time PCR detection, and analyze the results. 2) Key steps include setting up a two-step PCR protocol with 40 cycles, running an auto-gain optimization, starting the PCR run using the template, and analyzing the run to calculate threshold cycles and export quantification results to Excel. 3) Analyzing the run involves manually defining the baseline and threshold, and exporting the quantification cycle (Ct) values for each well to Excel for further analysis.

1. Technical Note
QIAGEN Rotor Gene Q: Software V2.0
Instrument Setup Instructions for RT2 Profiler™ PCR
Arrays
Preparation
Before the Experiment (Presetting the Machine will save time for your run):
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Please make note of the Rotor-Gene Q Series software version on your instrument.
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For more information, please click the help button or refer to the Rotor-Gene Q Manual available at
http://www.qiagen.com.
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Set up a PCR protocol template file on the Rotor-Gene Q Series software as follows:
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Open the Rotor-Gene Q Series Software 2.0 on the desktop of the computer that is connected to the
Rotor-Gene Q.
Select File ► New. The New Run dialog box will appear (Note: the New Run dialog box may open
automatically).
Under the Advanced tab, select Two Step.
Click New.
Under the Welcome to the Advanced Run Wizard! tab, select Rotor-Disc 100.
Ensure locking ring has been attached to the Rotor-Disc 100 and check Locking Ring Attached
box.
Click Next.
Under the Miscellaneous Options tab, set Reaction Volume (µl) to 20.
Click Next.
Click the Edit Profile tab.
Under the Edit Profile tab (Figure 1), adjust parameters to reflect the following:
Hold
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Hold Temperature: 95°
C
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Hold Time: 10 mins 0 secs
Cycling
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This cycle repeats 40 time(s)
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95° , 15 seconds, Not Acquiring
C
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60° , 30 seconds, Acquiring to Cycling A on Green
C
Click Insert After ► New Melt. Ensure Optimize gain before melt on all tubes is checked.
Click Ok.
Click Gain Optimisation.
Under the Auto-Gain Optimisation Setup tab, click Optimise Acquiring.
Click Ok.
Ensure Perform Optimisation Before 1st Acquistion is checked.
Click Close.
Click Next.
Click Save Template and enter RT2_Rotor_Gene_Q as the template name.
Click Save.
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2. Technical Note
Figure 1
Edit Profile Tab
Performing Real-Time PCR Detection
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If the Rotor-Gene Q is off, switch on the instrument, and ensure the standby light is lit.
Open the Rotor-Gene Q Series Software 2.0.
Under the New Run dialog box, click on the Quick Start tab, and select Open a Template In Another
Folder.
Click New.
Locate RT2_Rotor_Gene_Q Template file.
Click Open.
Under the 1. Rotor Selection tab, select Rotor-Disc 100.
Ensure locking ring has been attached to the Rotor-Disc 100 and check Locking Ring Attached
box.
Click Next.
Under 2. Confirm Profile tab, verify desired profile.
Click Start Run.
Enter name for run and click Save.
Rotor-Gene Q run will now commence.
After the PCR Run
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Once the PCR run is complete, observe the Sample Bank (right side of screen, Figure 2).
Click Bank On.
Click All On.
Select Analysis in program bar.
Under Quantitation tab, select Cycling A. Green.
Click Show.
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3. Technical Note
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Calculate the threshold cycle (Ct) for each well using the instrument’s software.
a. To define the Baseline (Figure 1):
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Observe amplification plots in Linear View.
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Select Dynamic Tube (default analysis setting) to ensure the average background of
each well is determined just before amplification commences.
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(Optional) Select Ignore First. Fluorescent signal from the initial cycles may not be
representative of the remainder of the run. Thus, better results may be achieved if the
initial cycles are ignored. Up to 5 cycles can be ignored.
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(Optional) Select Noise Slope Correction. Selection of this option can improve data
whose baseline (initial cycles) is noticeably sloped. Noise Slope Correction improves
the data when raw data backgrounds are observed to slope upward or downward
before the takeoff point (Ct).
*IMPORTANT: Ensure that all selections remain consistent across all PCR Array runs in the same analysis.
Figure 2
Setting the Baseline
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Manually define the Threshold Value (Figure 3):
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Observe the Log View of the amplification plots.
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In the Ct Calculation box (under Sample Bank) click the button beside the Threshold
box.
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Move mouse to Amplification plot and click mouse to place threshold above the
background signal but within the lower one-third to lower one-half of the linear phase of
the amplification plot.
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Right click on Quant. Results window.
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Click Export to Excel.
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4. Technical Note
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This file format can be opened in the Microsoft Excel Program.
Figure 4 illustrates the layout of the Rotor-Gene Q analysis window.
Figure 3
Setting the Threshold
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5. Technical Note
Figure 4
Rotor-Gene Q Analysis Window
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