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HRM instrumentaion

any suggestions ??

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4 replies to this topic

#1 nightingale

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Posted 06 June 2014 - 03:30 AM

Dear all,

 

We are to buy a real-time PCR device, with HRM technology ...

From your experiences, do you recommend any specific device ???

 

Many thanks


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#2 Trof

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Posted 06 June 2014 - 06:59 AM

If you are serious with HRM, probably only Rotorgene machine.

It's air-heated which ensures quick and uniform(er) temperature changes, that are necessary for HRM.

 

Roche claims their LightCycler 480 (block-based) has more temperature uniformity than other block cyclers, but in my oppinion it's still not ideal. We have it and for routine HRM it's not very robust. I think that would be similar for other block-based cyclers (ABI etc.).

 

Here is a page with many resources about HRM (and qPCR in general) including instrumentation.

http://www.gene-quan...ion.de/hrm.html

 

The described problem with temperature shifting is what I observed with our LC480. Problem is, there are many types of SNPs (4 general classes) and each one can be more or less difficult to identify by HRM, also methylation is something differen etc.

 

Rotorgene 6000 was considered the best at time (some time ago, I'm not sure where it gone now, as I learned Rotorgene was buyed by Qiagen and renamed), since in principle the air-heating is more rapid and accurate. But also less comforable to pipet and for the comsumption of capillaries. Also fits less samples at once. But as a golden standard for HRM, it had also better analysis software. HRM analysis module is bought separately in case of LightCycler and I think needs a special sotware alone in case of ABI cyclers.

 

I would say, if your primary goal is the best HRM, go for Rotorgene, if you need more universal cycler, that is block-based for 96 or 384 wells, try Roche LightCycler 480.


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#3 nightingale

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Posted 06 June 2014 - 07:52 AM

Grateful to your precious notes Trot , I am familiar with the link you kindly refered to ...

Our main aim is HRM yes , so after contacting rotorgene , this is what they offered : rotor gene q PCR machine.

Here I attach the document they sent.

 

What you kindly think ??? Will go for it ???

 

 

In your post, you said consumption of capillaries ... what do you mean by this kindly ????

 

 

Grateful ...

Attached Files


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#4 Trof

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Posted 06 June 2014 - 01:59 PM

Yes, I think that is the new version of Rotorgene now. You can read the specifications yourself. I don't have a personal experience with HRM on this one, so I can't tell you more.

 

And for the capillaries.. air-heated cyclers don't use classic 0.2 ml PCR tubes or plates, but instead needed a special thin wells called "capillaries" that were usually more expensive than classical wells, but as I look now, they seem to have different variants of shapes and numbers per run. So you should also let them inform you about the cost of consumables for the cycler. As they may be non-standard and even need a non-standard pipetting stands and other acessories.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 nightingale

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Posted 06 June 2014 - 10:50 PM

i got your point, thank you. 


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