I have been using Corbett Rotorgene 6000 for HRM analysis. In my new lab, we have BioRad CFX96 machine but its software (Precision melt analysis) is very expensive (5000 USD) unlike rotorgene which uses the same software for HRM analysis as used for other Real time data analysis. Is there any alternative of buying 5000 dollar software for CFX96 for performing HRM analysis?
Also, we do have Applied Biosystems® 7500 Real-Time PCR Systems. I don't know if this can be used for HRM analysis with similar/useful sensitivities as Rotorgene machine. Any help in this regard would be highly appreciated.
Please also comment if ABI 7500 real time system also requires a separate software for HRM analysis (like CFX96 system) or it can be done with the software used for routine real time data analysis software.
Hi, I am afraid I can´t be of great help but I can tell you this: We have many different qPCR machines and none is as good as our RotorGene for HRM. I fear you will get disappointed what ever solution you come up with since you are already used to the best machine.
Saying this I nevertheless hope I am wrong and wish you all the best.
AB7500 can be used for HRM, but you will need a special software for this I am afraid. In the UK the price is ~2K GBP. Also you will need a special calibration plate which you need to use every 6 months. It's quite good for HRM, though. Sensitive and reproducible. Still, I would prefer RotorGene for HRM for this particular application.
Thanks Maxim Freidin for your suggestion. Is it really compulsory to use that calibration plate? If someone doesn't have that plate but still get melt curve data and analyse data using appropriate software (for AB7500), would that be meaningful? What would be the difference in melting data if calibration is not done compared to that with calibration? Thanks and waiting your reply.
You do need calibration data to run the HRM software for AB7500. It doesn't work without it, I am afraid. It's compulsory. You also need to calibrate AB7500 for the specific dye. This data are specific for a particular machine. Formally, you can copy these data from another computer where the software installed, but HRM is very sensitive approach, so your results won't be precise.
Here are some links you may find useful
https://tools.thermofisher.com/content/sfs/manuals/cms_090394.pdf
https://www.thermofisher.com/uk/en/home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/meltdoctor-hrm-reagents.html
http://www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=93401&filterDispName=High+Resolution+Melting+%28HRM%29+Analysis&filterType=1&OP=filter&filter=ft_1701%2Ff_2020770*&_bcs_=H4sIAAAAAAAAAI1P0WrDMAz8Gr8srDhx2bLHLqWldIxB2N6FrTYBOy62spC%2F36VQ6FMZiDvpfMin%0A51Lp%2BitFN1rJhapeipbTb285P9A7kYsyG1XtUNM0raTjFOKpz%2BCVjQHymAE8ALoYGBSTw2MnwWOD%0AqsxSupY08jLrVw0qdQnnm1nr8gljKzQ4Sg6rmuKDHBZcs1DA9%2B9FEwdJ0f8vkdnU%2BpYCbEnIx%2FMt%0A43cLINcntoLOizI7G5wy28PP%2FhPmbZ8vnuaGhM8xzQgB8cgzHNe49zedyOflqD%2FygQxIXAEAAA%3D%3D
Dear Maxim Freidin, do you have any rough idea of the price of Calibration kit for ABI 75000 system? It is taking sometime for the local supplier to give this estimate. Thanks.
In the UK, list price for 96-plate is £492. You can also get a tube to prepare the plate yourself, the price is £396. No idea how much it is in your place. You can get the price on Thermo Fischer web site specifying your location.
I think reading this paper may be of some help to you. They discuss different instruments and software for HRM analysis.
Li, M., Zhou, L., Palais, R.A., Wittwer, C.T., 2014. Genotyping accuracy of high-resolution DNA melting instruments. Clinical Chemistry 60, 864–872. doi:10.1373/clinchem.2013.220160
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