Standardization of HRM for methylation analysis - (Feb/21/2011 )
I am trying to standardize HRM for methylation since last few months but have not succeeded yet. the problem that i am facing is that there is hardly any difference in melting in 100% methylation standard compared to 0% methylation standard. even mixed standards with different methylation %ages and unknown patient samples also don't show any difference in melting. all these standards and unknown samples show melting around 76C.
I am using LightCycler480 from roche and their HRM dye for this technique.
the conditions that i am using are as under:
54C annealing temperature, 3mM MgCl2, 0.15 micromolar primer concentration.
Kindly suggest me some changes needed to be done to get the proper shift in melting temperature for different standard
your positive approach will be highly appreciated.
with regards
beenu
Hello everyone
kindly suggest some changes that can be done to get proper result and proper melting.
i am not able to get some shift in temperature for melting of different standards.
kindly help.
with regards
beenu
beenu on Tue Feb 22 05:51:36 2011 said:
I am trying to standardize HRM for methylation since last few months but have not succeeded yet. the problem that i am facing is that there is hardly any difference in melting in 100% methylation standard compared to 0% methylation standard. even mixed standards with different methylation %ages and unknown patient samples also don't show any difference in melting. all these standards and unknown samples show melting around 76C.
I am using LightCycler480 from roche and their HRM dye for this technique.
the conditions that i am using are as under:
54C annealing temperature, 3mM MgCl2, 0.15 micromolar primer concentration.
Kindly suggest me some changes needed to be done to get the proper shift in melting temperature for different standard
your positive approach will be highly appreciated.
with regards
beenu
HRM can be tricky, especially on LC480. I didn't respond, because I neved did methylation HRM, but you can just try the general recomendations.
Have your set of standards (maybe not all of them just select like 100%, 50% an 0%) and run all possible variants of the reaction and see which condition distinguish your standards best. You didn't write what did you tried and what not.
Did you try all the recomended concentration of MgCl2? Did you tried different annealing temperatures for your reactions? Did you check melting in the Tm call module to see if there aren't any primer dimers? Did you tried to adjust HRM range? How long is your product? How many methylation sites do you have in the amplicon?
You're talking about Tm, but that's not that important, important is mostly the shape of the melting curve. Are you doing the analysis in Gene scanning mode? Because there are no Tms, actually the program makes by default a temperature shift of all the curves, so you won't see a difference.
Try to post some images of your curves, so we can see them.
I have tried MgCl2 concentrations ranging from 2.5-4 mM and temperatures from 52-60 but there is no shift in temperatures for a particular genes for 100% and 0% methylation standards. it's amplicon is 132bp long with CpG sites. there are no primer dimers in this gene amplification that i have checked from Tm calling mode. in gene scanning mod there is hardly any change in shape of the curves.(CASE 1)
More over i am facing the problem of nonspecific amplification for few other genes , as a result i am not able to distinguish between specific and nonspecific Tm in Tm call mode for standards as well as samples.
Recently i have got temperature shift for standards in case of 2 genes (for 1 there is 6 degree difference (CASE 2) but for other one there is 1 degree difference(CASE 3) ) more over both have some nonspecific amplification . so i can t get a proper normalized curves on gene scanning mode.
here i am attaching pics of all the 3 cases . kindly help me to remove these issues of no shift or lesser shift in Tm and nonspecific amplification .
With Regards
Beenu
Trof on Sun Mar 6 15:07:12 2011 said:
HRM can be tricky, especially on LC480. I didn't respond, because I neved did methylation HRM, but you can just try the general recomendations.
Have your set of standards (maybe not all of them just select like 100%, 50% an 0%) and run all possible variants of the reaction and see which condition distinguish your standards best. You didn't write what did you tried and what not.
Did you try all the recomended concentration of MgCl2? Did you tried different annealing temperatures for your reactions? Did you check melting in the Tm call module to see if there aren't any primer dimers? Did you tried to adjust HRM range? How long is your product? How many methylation sites do you have in the amplicon?
You're talking about Tm, but that's not that important, important is mostly the shape of the melting curve. Are you doing the analysis in Gene scanning mode? Because there are no Tms, actually the program makes by default a temperature shift of all the curves, so you won't see a difference.
Try to post some images of your curves, so we can see them.
Using only 0% and 100% methylated sample is no good. They only differ in Tm and that can be a slight change in the unoptimized reaction, especially in the case of single methylation site (you didn't write how many CpGs are in your amplicon). 50% methylated creates heteroduplex, that form distinctive shape in Gene Scanning. Did you follow the manual on how much template and primers to try in the reaction?
The other two images look like lousy specifity, you need to get specific peak first before you try to analyse melting.
Also, don't write comments on my profile only to get my attention, I'll respond when I'll have a spare time.