Troubleshooting Unexpected Results with the Infinium HD FFPE QC Assay Protocol

The Infinium HD FFPE QC Assay Protocol is used to determine whether DNA samples are suitable candidates for the Infinium HD FFPE Restore Protocol which is required for both the Infinium HD FFPE Genotyping Assay and the Infinium HD FFPE Methylation Assay.

If unexpected results are obtained, consider the following troubleshooting recommendations:

Verify DNA Quantification Method

• Confirm that samples are quantified using a fluorimetric method (eg, Qubit or PicoGreen).

• See the Preparation section of the protocol.

Verify Thermocycler Settings

• Confirm that the thermocycler settings are specific to the qPCR master mix used.

• Refer to Step 11 of the protocol. Depending on the master mix manufacturer's recommendations, the following step(s) may be required:

• • • 50°C for 2 minutes and/or

      • 95°C for 10 minutes

Verify qPCR Analysis Settings

• In this protocol, Ct (threshhold cycle) is referred to as Cq (quantification cycle).

• Confirm that the qPCR run is configured to perform the correct analysis. Refer to Analyzing Data, Step 4 of the protocol.

  • Analysis must subtract average Cq value of each sample from average Cq value of the QCT_ST control sample.

  • Analysis must not quantify Cq values against a standard curve.

  • If necessary, consult the qPCR instrument or software manufacturer for assistance with configuring the run and analysis settings correctly.

Data Interpretation

• The following results are considered acceptable.

  • Negative Delta Cq (ΔCq) values for the samples which indicate that the sample DNA is more concentrated than the QCT_ST standard sample

  • The NTC (no template control) Cq is > 10 cycles than the QCT_ST Cq values.

  • Samples with ΔCq values < 5, including negative ΔCq values, are considered good candidates for restoration.

References:

Infinium HD FFPE QC Assay Protocol

Infinium HD FFPE Restore Protocol

For any feedback or questions regarding this article (Illumina Knowledge Article #7905), contact Illumina Technical Support [e-mail].