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General

Yes, the instrument should be calibrated for each HRM dye, and the data analysis should be done with the matching calibration file.

No. We do not provide primer design service for HRM.

The HRM calibration is a 3-step procedure. In some instrument software the calibration process has been streamlined to a single-step procedure (applies to the ViiA™ 7 Real-Time PCR System, QuantStudio® 6 and 7 Real-Time PCR Systems, and the QuantStudio® 12K Flex Real-Time PCR System).

For the HRM calibration, the first step is a PCR reaction to generate the HRM dye plate. The reaction plate contains a DNA template, a primer pair, and the PCR mix with HRM dye. The second step is dye calibration using the PCR plate from step 1. The last step is a melt curve run, again using the same plate. For details, please refer to the HRM guides for each instrument: 7500 Fast Real-Time PCR System (with software version 1.4 or 2.0.4 and below), 7500 Fast Real-Time PCR System (with software version 2.0.5 and above), 7900HT Real-Time PCR System, StepOne™ and StepOnePlus™ Real-Time PCR Systems, ViiA™ 7 Real-Time PCR System, QuantStudio® 6 and 7 Real-Time PCR Systems, and QuantStudio® 12K Flex Real-Time PCR System.

You can order a synthetic positive control plasmid using our GeneArt® Gene Synthesis services. The final product is sequence verified and provided as 5 µg of lyophilized plasmid. Alternatively, you may be able to find a positive control sample from Coriell.

Software Data Analysis

For the 7500 Fast Real-Time PCR System, if the software version is below v2.0.4, the compatible HRM Software is v2.0.1. If the 7500 Fast Real-Time PCR System has been upgraded to software version 2.0.4 or above, then you will need to get HRM Software version 3.0.1.

There are a few possibilities. First, make sure the HRM Software version is v2.0.1, and the 7900HT Fast Real-Time PCR System software version is v2.3 or above. Second, check that the run method used was as recommended in the HRM protocol; make sure the ramp rate for the dissociation stage is 1%. Then try to open the calibration file from the HRM Software; if it does not open, the calibration file is defective. The defects could be due to a bad calibration plate or instrument uniformity issue.

Check that there are not outliers on the plate. You cannot omit any wells on the HRM calibration plate.

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