Difference between revisions of "QPCR Analysis on ThermoCloud"
From Bridges Lab Protocols
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**Verify that each of your replicates amplified for each target. | **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'''. | **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 the above criteria by clicking the well not the plate schematic and '''Omit''' under the '''Action''' dropdown box above the plate schematic. | + | **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. | *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. | *Click '''Next''' and '''Export''' your results to the appropriate project folder. | ||
Latest revision as of 16:49, 4 December 2018
- 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.
