Troubleshooting the Infinium HD FFPE QC Assay Protocol
The Infinium HD FFPE QC Assay Protocol determines whether DNA samples are good candidates for the Infinium HD FFPE Restore Protocol.
With unexpected results, consider these troubleshooting tips:
Confirm that samples are quantified using a fluorometric method (e.g., Qubit or PicoGreen).
See the Preparation section of the protocol.
Confirm that the thermocycler settings are specific to the qPCR master mix used.
See Step 11 of the protocol: - 50C* for 2 minutes and/or 95C* for 10 minutes are included *if required by the Master Mix manufacturer.
In this protocol, Ct (threshhold cycle) is referred to as Cq (quantification cycle).
Confirm that the qPCR run is set to perform the correct analysis.
See Analyzing Data step 4 of the protocol.
Analysis must subtract average Cq values of samples from average Cq values of the QCT_ST sample.
Analysis must not quantify Cq values against a standard curve.
If needed, consult the qPCR system/software manufacturer for support on how to set up the run/analysis correctly.
It is acceptable to have negative Delta Cq values for the samples. This result means that the DNA is more concentrated than the QCT_ST standard sample.
Importantly, the data is acceptable if the the NTC (no template control) Cq is > 10 cycles after the QCT_ST Cq values.
Samples with Delta Cq values < 5 (including negative Delta Cq values) are good candidates for restoration.
For any feedback or questions regarding this article (Illumina Knowledge Article #7905), contact Illumina Technical Support techsupport@illumina.com. |
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