High Resolution Melt (HRM) Support—Getting Started

Among our real-time PCR instruments, the following are compatible with HRM:

• 7500 Fast Real-Time PCR System
• 7900HT Fast Real-Time PCR System
• StepOne and StepOnePlus Real-Time PCR Systems
• ViiA 7 Real-Time PCR System
• QuantStudio 6 and 7 Pro Real-Time PCR Systems
• QuantStudio 6 and 7 Flex Real-Time PCR Systems
• QuantStudio 12K Flex Real-Time PCR System
• QuantStudio 1 Real-Time PCR System
• QuantStudio 3 Real-Time PCR System
• QuantStudio 5 Real-Time PCR System

The instrument will need to be calibrated for HRM. This includes an HRM dye calibration and a melt curve file for data analysis. You can find the appropriate HRM calibration plate for your instrument here. For detailed calibration instructions, please refer to the HRM guides for each instrument and the appropriate software:

QuantStudio™ 3 and 5
QuantStudio™ 12K Flex
QuantStudio™ 6 and 7
ViiA™ 7
7900 and 7500 SDS v1.x

We provide the MeltDoctor™ HRM dye for HRM applications. Other commonly used dyes for HRM include LCGreen® Plus, Eva Green® dye, and SYBR® Green dye.

The PCR primers need to be designed for robust performance and must be specific to the region of interest with a minimum of primer-dimers and nonspecific products. A variety of software tools are available to help design HRM primers, including Primer Express® 3.0 Software, Primer3, Primer3Plus, and Methyl Primer Express® Software for methylation-sensitive HRM analysis.

We have two options for HRM reagents. The MeltDoctor™ HRM Master Mix contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets. It is recommended for most HRM application except methylation-sensitive HRM. The MeltDoctor™ HRM Reagent Kit provides individually packaged PCR components and the MeltDoctor™ HRM Dye required for high-resolution melt analysis that is recommended for methylation-sensitive HRM analysis (as it does not contain dUTP).

Yes, if you are using HRM for genotyping, you can use our MeltDoctor™ HRM Positive Control Kit to help troubleshoot your experiment. The kit contains control gDNA for each allele (A, A/G, and G), along with a HRM Primer Mix to match.

We do not have any HRM calibration plates for 96-well standard blocks. To perform HRM experiments on such a system, you can instead use our MeltDoctor™ HRM Calibration Standard to create your own calibration plate.

You can use HRM to discover novel SNPs over a specific target region, or to quickly screen samples for a specific target SNP. In both cases, you can track the mutation changes by viewing the aligned melt curves.

If you are using the QuantStudio 3/QuantStudio 5 Real-Time PCR Systems, 7500 Real-Time PCR System (with SDS v2.0.5 or later), or StepOne/StepOnePlus Real Time PCR Systems, you can purchase High Resolution Melt (HRM) Software v3.1. Once activated, this software will not require annual renewal. If you have the older HRM software v3.0.1 (for use with StepOne Plus, StepOne or the 7500 Fast (SDS v2.0.5 or later) Real-Time PCR System, it will require license renewal on an annual basis. For use on the 7900HT Fast Real-Time PCR System, please use HRM Software v2.0.1. This software will require license renewal on an annual basis. When using either the ViiA 7, QuantStudio 6/QuantStudio 7 or QuantStudio 12K Flex Real-Time PCR Systems, the HRM software is already included in the instrument's application software, but requires activation of the HRM module within the instrument software by purchase of the license. This license requires an annual renewal.

We recommend using Continuous mode (set to 0.025 degrees C/sec) for HRM experiments. You can also choose “Number of Data Points per Degree”, but if you do so, make sure to collect at least 10 points per degree so that there is enough data for analysis. When in Continuous mode, the instrument uses a fixed ramp rate for the melt step and captures as many data points as possible within the time permitted. When in Data Point Per Degree mode, the instrument adjusts the step and hold for the melt step to achieve the specified number of data points.