Hi I use the same machine for HRM and is compulsory to use calibration kit for the correct calibration 

Hi I use the same machine for HRM and is compulsory to use calibration kit for the correct calibration.

This calibration is necessary for a correct use of precision analyses software that after the generation of the melting curves gives different clusters of memberships.

The generation of cluster that are different from wild type and mutated one is possible only with this calibration.

In your case I think that you can skip this passage because if i understood well you have to assess a dye.

In this case I suppose that you have to do only a melting curve after you PCR that you can appreciate without melt software that gives you also different cluster that in this case you don t need.

in file attached you can see an example of generation of cluster with Melt software 

I hope that I m clear

Ask if you have more question 

Daniela Cangiano,PhD

Dear Daniela Cangiano,

Thanks for your detailed answer. Actually I don't have enough funds at the moment to buy Precision melt software and kit. Therefore I just want to know that if I perform melt curve (using suitable dye) followed by PCR on CFX96 to obtain .prcl file, and then, later perform the clustering/genotyping using precision melt software with the help of someone else who have access to this software. Is it possible? Will you be able to help me to analyse my such data? Also, please suggest which dye is suitable for using with CFX96 machine. On rotorgene, I used Syto9.

Thanks for your help.

Regards,

Shahid

Dear Muhammad Shahid,

you are right the software is very expensive.

The mix that I use to perform the experiment is from Biorad and is named SSoFast mix with evagreen and you  can find the link below:

http://www.bio-rad.com/it-it/product/ssofast-evagreen-supermixes

Inside this mix there is Evagreen that works very well for HRM.

There is an other mix from Biorad  named precision melt supermix that they do properly for HRM:

http://www.bio-rad.com/it-it/product/precision-melt-supermix?pcp_loc=catprod

I never use the last one but seems works very well and maybe is more expensive than the other one.

The first mix  I use also  for simple real time PCR that i perform when I have to sequence some products of PCR.

I think that the main differences beetween Syto9 is the adsorbance and emission of fluorescence that in the case of EVAgreen is similar to FAM but sorry I don t know exactly for Syto9.

Of course I can help you with results but the most important think is that the file of PCR  that you obtain with CFX96 machine is with the fomat  .pcrd.

Precision melting software recognizes this format and generates different clusters for genotyping.

I hope that I m clear 

Best regards

Daniela Cangiano,PhD

Dear Daniela. Thanks for your above suggestions.

Is it really compulsory to use that calibration kit? If someone doesn't have that kit but still get melt curve data and analyse data using appropriate software, would that be meaningful? What would be the difference in melting data and its interpretation if calibration is not done compared to that with calibration? Thanks and waiting your reply.

TL;DR

• You cannot calibrate with a standard PCR reaction.
• You cannot analyze your melt-curves with Precision-melt software if the melting-curves were obtained from a machine that has not been calibrated
• If the calibration has not been done, the software will not be able to compensate for machine inconsistencies, reducing the precision of the curves
• An alternative for you would be to use this: https://github.com/liuyigh/PyHRM

The calibration kit contains a reaction which will allow the software to compensate for hardware inconsistencies, and the Precision Melt Software needs the calibration file in order to function properly (it will not even let you open a .pcrd file otherwise).

So, if you don't have the calibration file for the specific machine that you used to collect the melting-curves, you will not be able to analyze using the Precision Melt Software.

However, if you don't need the high precision and only want to detect melting differences that are relatively big, you can use this Python-script written by Yi Liu:

https://github.com/liuyigh/PyHRM

(original thread= https://www.researchgate.net/post/Melting_curve_analysis_software )

The script is surprisingly powerful and will even cluster your samples to the controls you load. You will however need some SUPER BASIC knowledge of scripting in order to get it to run. Good luck!

I am aware that this answer might be coming a bit late, but for future reference it might still be good to finally close the subject.

https://github.com/liuyigh/PyHRM

Hello all. For some reasons I have lost the calibration file! I can see my previous HRM experiments data on the precision melt software but when I want to import any new data, problem pops up! The error message is there is no calibration data. Can anyone borrow my a calibration file just to see if it can help? My machine is the BioRad C1000. Thank you.

Hello. I agree with Charbel Antoine Kreidy. HRM analysis is possible with custom software/pipelines (e.g. Article Genotyping Accuracy of High-Resolution DNA Melting Instruments

@ Muhammad Akbar Shahid hello, i meet the same problem as you. It is expensive for us to buy HRM calibration kit and HRM analysis solfware for CFX96 Real Time machine . How is it going with your HRM data?

Lin Dongfeng, you can easily perform HRM analysis in R as we did in a recent study: Article High-Resolution Melting (HRM) Curve Assay for the Identifica...

A description of what we did in R is below but let me start with something else first. We have used both, commercial software as well as R. Both returned the same results. Generally, I recommend using open source software rather than commercial software. I also argue that a 'calibration' of your machine is completely unnecessary and just a waste of money... simply run the intervals at which you increase the temperature at 0.1 or 0.2°C steps. Depending on your melting curves, you may consider using a 'next-generation' DNA-binding dye like EvaGreen instead of SYBR Green.

Here's what we did in R: "Relative fluorescence unit (RFU) data were obtained from CFX Maestro™ Software (Bio-Rad CFX Maestro 1.1 version 4.1.2433.1219) Biorad, Rüdigheim, Germany and analyzed in the R environment (version 3.6.1) [65]. RFU data were normalized by scaling all RFU values of each sample between 0 and 1. The negative first derivative (-d(RFU)/d(T)) was calculated employing the “diffQ2”-function in the R-package “MBmca” (version 0.0.3-5) and plotted to obtain normalized melting curves. The melting curves of the reference strains were generated from the mean fluorescence of the three technical replicates. Difference curves were obtained by subtracting the melting curve data of each reference strain or environmental sample from the mean melting curve data of all reference strains." (see Materials and Methods of the article above).

Cheers,

Lukas

@ Lukas Beule Great job! We are doing a qMSP experiment, and find it challenging to identify PCR amplicon than SNP. It is the best way to leave the the professional matter to the professional person. However, HRM analysis softwares are controled in several qPCR machine suppliers.

I had learned a little python before, but never get in touch with R. Do you have put your script on the github?

Really appreciate for your help.

Lin Dongfeng, I currently don't have the R script on GitHub. I would have to re-write it so that others can use it for their data. Anyway, I'll sent you a message and we can try to work things out!

Hi all,

I am glad to announce that you can now plot denaturation, melting and difference curves in R using our HRM.curve package (https://CRAN.R-project.org/package=HRM.curve). It only requires the raw RFU data of the melting curves that you can obtain from your CFX manager software or equivalent. Before you try it, please see the package description, the examples as well as our example dataset (https://github.com/LukasBeule/HRM/blob/main/HRM_example_data.txt). We tried to write the functions as intuitive as possible; however, if you have any questions, I will be happy to answer them. If you detect bugs or need additional features, feel free to contact me by email (my email address can be found in the package description).

-- Lukas