Dear all,
I wanted to run HRM ( High Resolution Melting) using Corbett Rotor-Gene 6000 Real Time PCR Machine. When I opened the HRM folder of software , there was two option for running including HRM and HRM with pre-amplification.
What is the difference between them?
Many thanks
F.Esmaeili
Dear Dr. Esmaeili
HRM analysis is performed on double-stranded DNA samples. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. In the sample tube, there are now many copies of the DNA region of interest. This region that is amplified is known as the amplicon. After the PCR process, the HRM analysis begins. The process is simply precise warming of the amplicon DNA from around 50 ˚C up to around 95 ˚C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart.
As I worked with that tool (Corbett) in NIGEB, the given functions are set up based on the type of your template samples. If you use gDNA, for providing more templates for HRM you should perform an extra amplification. While if you use PCR products, the pre-amplification of samples will not be required. You also have another accurate tool in NIGEB through which you can solve your problem. The MIC apparatus on the third floor of NIGEB can provide you an excellent experience with HRM analysis. I highly recommend you to use it. You can ask Dr. Mazinani or Mr. VatanKhah to manage a time for you to use the MIC apparatus.
Good luck
Dear Dr. Rasouli H.
Thank you for spending your time to write it all down. I really appreciate for your kindly help.
In fact, I used cDNA as an example to set up the reaction , and the HRM reaction was run with both options. Although the sample was cDNA and no per-amplification was performed, the result of HRM option was significantly better.
Best regards,
Dear Dr. Fazileh Esmaeili
I hope the answer supports your idea. In fact, HRM technique's quality depends on the quality of your genomic samples. For gDNA-based HRM analyses, the pre-amplification step is necessary. Please note that the interpretation of HRM outputs will require enough time and skill to find the best results for possible comparison of your genotypes. If you use plant-based genomic samples, please carefully read the available literature. For medicinal purposes, the interpretation of HRM results is easy and many studies simplified its procedures. Let me know if there are further questions.
Regards,
Rasouli. H., SWRI, Karaj, Iran.
Hi there
As my friend (Dr. Sharafi) has worked lots via PCR, he might be able to guide you in this matter.
Here is the whats app link : +98 914 980 3318
Best regards
Farzad Milan
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