QIAGEN Rotor Gene Q: Software V2.0 Instrument Setup Instructions for RT 2 Profiler PCR Arrays Preparation Before the Experiment (Presetting the Machine will save time for your run): Please make note of the Rotor-Gene Q Series software version on your instrument. For more information, please click the help button or refer to the Rotor-Gene Q Manual available at http://www.qiagen.com. Set up a PCR protocol template file on the Rotor-Gene Q Series software as follows: 1. Open the Rotor-Gene Q Series Software 2.0 on the desktop of the computer that is connected to the Rotor-Gene Q. 2. Select File New. The New Run dialog box will appear (Note: the New Run dialog box may open automatically). 3. Under the Advanced tab, select Two Step. 4. Click New. 5. Under the Welcome to the Advanced Run Wizard! tab, select Rotor-Disc 100. 6. Ensure locking ring has been attached to the Rotor-Disc 100 and check Locking Ring Attached 7. Click Next. 8. Under the Miscellaneous Options tab, set Reaction Volume (µl) to 20. 9. Click Next. 10. Click the Edit Profile tab. 11. Under the Edit Profile tab (Figure 1), adjust parameters to reflect the following: Hold Hold Temperature: 95 C Hold Time: 10 mins 0 secs Cycling This cycle repeats 40 time(s) 95 C, 15 seconds, Not Acquiring 60 C, 30 seconds, Acquiring to Cycling A on Green Click Insert After New Melt. Ensure Optimize gain before melt on all tubes is checked. Click Ok. 12. Click Gain Optimisation. 13. Under the Auto-Gain Optimisation Setup tab, click Optimise Acquiring. 14. Click Ok. 15. Ensure Perform Optimisation Before 1st Acquistion is checked. 16. Click Close. 17. Click Next. 18. Click Save Template and enter RT2_Rotor_Gene_Q as the template name. 19. Click Save. Page 1 of 5
Figure 1 Edit Profile Tab Performing Real-Time PCR Detection 1. If the Rotor-Gene Q is off, switch on the instrument, and ensure the standby light is lit. 2. Open the Rotor-Gene Q Series Software 2.0. 3. Under the New Run dialog box, click on the Quick Start tab, and select Open a Template In Another Folder. 4. Click New. 5. Locate RT2_Rotor_Gene_Q Template file. 6. Click Open. 7. Under the 1. Rotor Selection tab, select Rotor-Disc 100. 8. Ensure locking ring has been attached to the Rotor-Disc 100 and check Locking Ring Attached 9. Click Next. 10. Under 2. Confirm Profile tab, verify desired profile. 11. Click Start Run. 12. Enter name for run and click Save. 13. Rotor-Gene Q run will now commence. After the PCR Run 1. Once the PCR run is complete, observe the Sample Bank (right side of screen, Figure 2). 2. Click Bank On. 3. Click All On. 4. Select Analysis in program bar. 5. Under Quantitation tab, select Cycling A. Green. 6. Click Show. Page 2 of 5
7. Calculate the threshold cycle (C t) for each well using the instrument s software. a. To define the Baseline (Figure 1): i. Observe amplification plots in Linear View. ii. Select Dynamic Tube (default analysis setting) to ensure the average background of each well is determined just before amplification commences. iii. (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. iv. (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 (C t). *IMPORTANT: Ensure that all selections remain consistent across all PCR Array runs in the same analysis. Figure 2 Setting the Baseline b. Manually define the Threshold Value (Figure 3): i. Observe the Log View of the amplification plots. ii. In the Ct Calculation box (under Sample Bank) click the button beside the Threshold iii. 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. iv. Right click on Quant. Results window. v. Click Export to Excel. Page 3 of 5
vi. This file format can be opened in the Microsoft Excel Program. c. Figure 4 illustrates the layout of the Rotor-Gene Q analysis window. Figure 3 Setting the Threshold Page 4 of 5
Figure 4 Rotor-Gene Q Analysis Window Page 5 of 5