Can I use the CopyCaller Software if I am using a non-Applied Biosystems Real-Time PCR instrument?
Yes, although the data will not be directly compatible. You can use the example files that install with the CopyCaller Software as a template to use with your data. You will have to copy/paste in the sample/target names and Ct values before importing into CopyCaller Software.
My data will not import into the CopyCaller Software. What could be wrong?
In the instrument software, make sure that you selected Results from the export options. The data need to be set up as duplex reactions as well. If you ran the copy number assay and the reference assay in separate wells, the data will not be in the correct format.
CopyCaller Software gave the error "XXX.txt is not a compatible file and cannot be added to the analysis. Verify that the file format is compatible with the software." What can I do now?
Check your export results with the instrument software. The data columns need to be in the following order: Well, Sample Name, Target Name, Task Reporter, Quencher, CT. If needed, rearrange the columns and export the data again for CopyCaller Software.
My results are different from what I expected when working with your Copy Number Variation Assay. Is there something wrong with the assay?
Check that the reference assay is performing consistently across all samples. Check the concentration of your samples and make sure they are normalized. We also recommend you to use several copy number assays targeting the same variation to validate the data.
What can I do if the copy number confidence values are very low?
Confidence values can be low for several reasons. Check your data for the following:
- Large variability in ΔCt values across the plate.
- Low number of replicates per sample (recommendation is for at least 4 replicates per sample).
- Sample copy number is high (> 3). You may want to use copy number bins instead for high copy numbers. Also keep in mind that CopyCaller Software is best for copy ranges of 1-5. If you have higher copy numbers, such as with transfections/transductions, it is best to use a standard curve.