Recently a co-worker at my previous affiliation (VIAEP) ask me which model, which made of a Real-time PCR machine we should buy. Well, to be honest, I have no experience with real-time PCR yet, I then asked a groups of experts in the field, and here I summary their opinions after I conceived it.

Factors you need to consider:

Heating (Cooling)

• How is the sample heated? (cooled?)
• Is it evenly heated in all samples? (cooled?)
• How fast does heating occur? (cooling?) 

Fluoresence Detection:

• What are the excitation wavelengths?
• What are the emission wavelengths?
• How many dyes can be used in a single reaction?
• How many fluorescence acquisitions per cycle?
• How is detection performed after the run is complete, can you do melt-curves?
• What are the resolution capabilities of the instrument during a melt?  

Reporting

• Does your instrument have an integrated data analysis package?
• Can analysis be performed in real-time?
• Are the types of data analysis necessary for the project available with the instrument? 
• If not is there a ‘third-party’ software package that is available?
• Is the ‘third-party’ software updated regularly, and are upgrades compatible with upgrades to your instruments software?

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Consider these features carefully when purchasing a new instrument or when establishing the technical specifications for a new assay. Dr. Caplin suggests, “Spend a little more of your project dollars to get a real-time PCR machine that has all of the flexibility you need in the thermal cycling programming, the detection formats, AND in the data analysis.”.

Developing an assay?

According to experts even the simplest of goals often provoke frustrating questions that need to be answered at the start of any new project: 

1. What is the end-goal of the assay? Including limits of sensitivity and specificity, what should the real-time PCR assay do?
2. What is possible with the real-time PCR instrument in your lab?
3. What is feasible within a real-time PCR format? Does real-time PCR as a technology make sense for the project?

Opinions from experts replied to my e-mail: 

Dear All,

I have no previous experience with realtime PCR however, now I need to make a suugestion to my friends regarding which model, made of thermo cycler we should buy. I would appreciate if anyone out here could give me some specific advice. Our main purpose is to quantify transgenes in GMO derived food.Thank you very much in advance

Dzung Le

Replied by Mikael Kubista, TATAA Biocenter AB, Sweden

What instrument to chose depends very much on your throughput, whether you will purchase kits or setup your own assays, the kind of support you will need etc. Our centers offer QPCR training where you also get a chance to try the most common QPCR platforms and with expert guidance you will be able to decide yourself which instrument suits your needs the best. 

Replied by Pierre Lebel MD FRCP, McGill University Health Center, Montreal General Hospital, Montreal (Quebec) Canada

We are LightCycler 480 user but not so happy! We have add trouble to transfer FRET probes assays from LC1 to LC 480! The master mix sold with the LC 480 is not working. We have to use the LC1 master mix… and this is more expensive. We have also tried the Qiagen multiplex master mix: working fine but… not optimized for short cycles (OK for 60 seconds cycles (TAT 2h30) but no amplification with shorter cycling time (TAT 45 minutes).

Replied by Brian Erich Caplin, Ph.D, President of Fluoresentric, Inc.

There certainly are other factors to consider in choosing a real-time PCR instrument, than sample volume and number of samples:
You should consider the thermal cycling temperature uniformity, the suitability of the vendor’s software package for the specific sort of application you intend to perform, and the selection and number of dyes that you can use on the instrument. Ask a few questions of your sales reps..test a few out..learn about the machines before you buy. These are expensive machines and no one machine is the best for all applications…regardless of the opinions posted here. 

Shef suggested "Go for ABI7900 which takesonly 10 microliter sample and is 96 well platform", however, Mats disagreed "If you want to do reliable quantifications complex samples like GMO food you might get inhibiton problems if you use only 10 microliter samples." (PCR inhibition is a serious problem – Dung Le)

Duncan, a satisfied user of LightCycler 480 at GeneSys Ltd, suggested "a LightCycler 480 which will also do 10ul, is much faster cycling, a wider fluor range plus you can buy both 96 and 384 well blocks which take only 60 seconds to swap over."

I hope you know what to do now in order to get the right cycler that best suit your need!

Dung Le

Suggested further readings: Renewing the Promise of Real-time PCR