QPCR Analysis on ThermoCloud

• Log into Thermo Cloud (https://apps.thermofisher.com/apps/dashboard using the login information for your lab (the Bridges Lab account data is located on the side of the machine.)
• Click on the File Folder icon and find your folder (or the folder in which you saved your run).
• Select the file with extension .eds. The program will automatically take you to the Results tab of your run.
• Above the graph, use the dropdown box to select Melt Curve.
• Click on Target above the plate schematic to select one target at a time.
  • Verify that there is only one (prominent) peak for each of your targets.
  • Verify that each of your replicates amplified for each target.
  • If there is a target that yields 2+ peaks over all samples, mark that target as unusable in the Primer Database.
  • Exclude samples that did not pass the above criteria by clicking the well (not the plate schematic) and select Omit under the Action dropdown box above the plate schematic.
• Check the technical replicates by choosing the Table View and arranging them according to target. Verify that the CT values have <1.5 difference. Omit outliers.
• Click Next and Export your results to the appropriate project folder.
• Under App Connect on the right hand side click on Relative Quantification (qPCR). Open your run.
• Under the Overview tab, assign biological groups to each of your samples (if needed).
• If you need to omit samples from the analysis, do so under the Data Review tab by clicking on the affected target, highlighting, and omitting the wells with outliers.
• Under the Analysis tab click the blue Analyze button in the top right corner to visualize your adjusted data.
• Export your data to the appropriate location.