Technical Note Eppendorf Mastercycler ep realplex Instrument Setup Instructions for RT2Profiler™ PCR Arrays ®Preparation Before the Experiment (Presetting the Run Template will save time for your run): 1. Make sure the correct thermal block cycler (silver or aluminum) is in place. 2. Turn on the Mastercycler instrument; the main switch is located at the back of the instrument. Turn on the computer linked to the instrument and log on to Window XP. 3. Start realplex software version 2.2. 4. Enter Username and Password when the Login dialog box appears and click OK to proceed with the login. The Plate Layout window (Figure 1) will appear. Figure 1 Plate Layout Window Page 1 of 14 Technical Note Background Calibration 5. Before setting up an experiment, it is necessary to perform background calibration using an empty, capped A plate. Click on Setup, then Background Calibration. A list of previously performed calibrations will appear. Click New… to open the following dialog box (Figure 2): Figure 2 New Background Calibration Dialog Box 6. Enter SABiosciences A Plate as the name of the background plate. The default block and lid temperatures (60.0 and 105°C, respectively) do not need to be changed. Insert the background plate into the Mastercycler with well A1 at the upper left corner; slide the heated lid closed, pull the sealing clamp into the closed position and click Calibrate. Click OK to begin calibration. 7. After the first reading, the software will prompt you to turn the plate. Open the realplex instrument and turn the background plate so that well A1 is in the lower right corner. Then close the instrument and click OK to continue the calibration. 8. If background calibration was successful, the plate layout will be highlighted in green and a dialog box will appear with the message Calibration successfully finished. Click OK to continue with the experiment setup. **If background calibration fails in some positions, those wells will be highlighted in orange. Failure may be a result of fluorescence contamination on the plate or in some wells of the thermoblock. Retry calibration with a new A Plate; if this also fails, clean the thermoblock as instructed in the instrument manual.Page 2 of 14 Technical Note RT2 Profiler PCR Array Template Setup 9. In the Plate Layout window, specify the following setup parameters: Filter 520 nm: SYBR Sample Vol: 25 µl Probe: SYBR Green Background: SABiosciences A plate 10. Select all wells, then right-click in the blue area and select “Unknown” to complete plate layout (Figure 3). Figure 3 Complete Plate Layout Window Page 3 of 14 Technical Note 11. Select PCR Program in the navigator window at the upper left corner of the screen. The default PCR program will appear (Figure 4). Figure 4 PCR Program Window 212. Edit the default program to create an RTProfiler PCR Array template. Right-click in segment 2, 3, or 4, then click Delete. All three segments will be deleted. Page 4 of 14 Technical Note 13. Right click on END. Select “Add Step”, then select “2 Step Cycle” and click OK (Figure 5). Figure 5 Adding 2-Step Cycle Page 5 of 14 Technical Note 14. Right click on END. Select “Add Step”, then select “Melting Curve” and click OK (Figure 6). Figure 6 Adding the Melting Curve 15. Change the length of the heat activation step to 10 minutes. To do so, click on 02:00 in segment 1 and enter 10:00. The final parameters for the PCR program should be as below: Program Segment Hold (mm:ss) Cycles Temperature (°C) Heat Activation 1 95.0 10:00 1 2 95.0 00:15 PCR cycle 40 3 60.0 01:00 4 95.0 00:15 5 60.0 00:15 Melt Curve 1 6 20:00 7 95.0 00:15 Page 6 of 14 Technical Note Temperature Step Parameters 16. Double-click on the Page 7 of 14 Technical Note 17. When the PCR Program setup is complete, the window should appear as in Figure 8. Figure 8: Final PCR Program Screen 18. Click File and select Save As. In the Assay Name field enter the filename RT2ProfilerPCRArraySilver or RT2ProfilerPCRArrayAluminum depending on which sample block is being used. From the drop-down list for Assay Type, select Template. Click OK. Page 8 of 14 Technical Note Performing Real-Time PCR Detection To load the PCR Array plate and start the run: 1. When the PCR Array is prepared and centrifuged, bring the plate to the realplex instrument to load. 2. Make sure the correct thermal block cycler (silver or aluminum) is in place. 3. If the realplex instrument is not on, turn it on using the main switch at the back of the instrument. Turn on the computer linked to the instrument and log on to Windows XP. 4. Start realplex software version 2.2. 5. Enter Username and Password when the Login dialog box appears and click OK to proceed with the login. The Plate Layout window will appear. 6. Wait for the LED on the top of the instrument to turn green. Make sure the instrument has been warmed up for at least 15 minutes before starting an assay. 7. Push the sealing clamp back into the open position, then slide the heated lid back. Place the PCR Array plate on the thermoblock with well A1 in the upper left corner. 8. Slide the heated lid forward and pull the clamp down. 9. In the Plate Layout window, click File then Open. Select the previously saved RT2ProfilerPCRArraySilver or RT2ProfilerPCRArrayAluminum run template as appropriate, then click OK. 10. Click File, then Save as. Save the file under a new filename as an Assay. 11. Click the Page 9 of 14 Technical Note Data Analysis 1. Click File, then Open Assay (Figure 9) and find the run file to be analyzed. Alternatively, upon completion of a PCR experiment, a dialog box will appear prompting you to start analysis. Click Yes. Figure 9 Opening the Run File Note: It is possible to open a data file for analysis during an ongoing assay. A message will appear alerting you that the file will be opened for data analysis only. Click OK to view the assay in the analysis window. You will not be able to view plate layout or PCR program information for the completed assay in this mode. Figure 10: Analysis only Page 10 of 14 Technical Note 2. In the navigator window at the upper left corner, click on Analysis. The top panel (Figure 11) features a Type of Application combo box. Click on the button to save the file with results. To view a selection of samples, you can highlight the desired wells in the Sample/Analysis window or in the MiniPlate Layout at the lower left part of the screen. Figure 12: Quantification Analysis window Page 11 of 14 Technical Note Setting Threshold and Baseline: 4. The Threshold should be set at the same level for all of the PCR arrays in your experiment. The threshold should be set using the log view of your amplification data and set somewhere in the lower 1/3 of your amplification curves. A good indication that the threshold is set in the proper location is that the values of the PPC assays on catalogued arrays should be between 18 Ct and 22 Ct. 5. To set the Baseline, use the Automatic Baseline setting. FIGURE 13: LOG VIEW of Raw Data Threshold Exporting the Ct Results: 6. To export Ct values, right click in the Sample/Analysis window and select Save Results As. Export results in text format to view results as a spreadsheet. Click OK. When prompted, choose the file destination, filename, and type. Click Save to export. To export results from a selection of wells, highlight the desired wells in the Sample/Analysis window before right-clicking to save results. Under Include, click the radio button for Selected Samples before proceeding with the export. Figure 14: Exporting the Ct Results Page 12 of 14 Technical Note Note: You can also export the entire run file containing all setup information, raw data, and analyses as a *.asy file. This will allow you to open the file using the realplex software on another computer. To do so, go to File and Export. The dialog box Export assay to file will appear. Select the destination for the file and enter a filename. The file type should be realplex-Exchange (*.asy). Click Save to finish. To import a file, open the realplex software and create a new document by clicking on the Page 13 of 14 Technical Note Page 14 of 14