Tips to know when choosing a cycler

[Wheygurt]

Hi,

I am part of a committee in charge of buying a qPCR cycler for business incubator specialized in biotechnology. Our main criteria is versatility since many companies with different needs will use it. But, there also the price and reagents cost to consider.

We looked at Roche's LC480, ABI StepOnePlus and 7900HT, Eppendorf's Mastercycler realplex and Stratagene's Mx3005. My questions is, what do I need to know that is not written in their beautiful brochures to make a better choice? Basically I'm asking to all of you qPCR users what do you recommend and why? We're looking for a good and robust machine.

Thanks in advance.

Eric (etrottier@biolactis.com)

[Suzanne]

Hi Eric,
Thanks for this post- I think its a great idea for a topic. I am sure many people have experience with different instruments and can tell you what is critical to have, nice to have, and what you wish it didn't have. Be as specific as possible. For example, user-friendly software is important, yes, but what about it makes it easy or hard.

When we get enough feedback, I'll submit it as a blog for www.bitesizebio.com and the author will be the members of the BioTechniques forum.

Look forward to your replies!

Best,
Suzanne

[WISBiomed]

Eric,

Besides the personal preferences, I would suggest the follow three factors incorporated in the selection process.

-Warranty: most come with one year. some with two years. Important for those systems have lasers.

-Run time: newer designs have fast ramping mechanism so the user will spend less time for the same cycle.

-Flexibility of the plate format. For incubators, you may have different projects / groups using the same unit. some may have a 96 or 384 plate or even 0.2 ml tubes. A system can use different plates will be a plus.

[nonrad]

I would suggest one of the faster machines so more runs per day
Some questions to consider (yeah sorry, not exactly an answer but. . )
- what applications will the companies have? (now and perhaps in the future?). This may play a part in the chemistries required
- What is the current expertise of the people involved in real-time PCR? (may require a lot or nil tech support from company)
- multiplexing likely?

[mchlbrmn]

My department recently purchased a machine.
I agree run time is important, especially if you'll have many users.

Another point that I'm also sorry isn't an answer: Is well to well variability an issue? Some companies claim there machine is more accurate than rivals. One company ran a test run in their demo machine by putting in a plate with a duplicate reaction in every well. They ran the plate and, voila, every well's Cp was amazingly consistent, even the corner wells. It occurred to me later that this actually proves nothing. Some primer sets work well over a wide range of temperatures and they may have picked a set that could work perfectly plus or minus 3 degrees from the anneal temp they used, while another primer set you might run could be near the point where it will stop working efficiently, so a 0.5 degree well to well temp variation could have a major effect on PCR efficiency. I just thought I'd alert you to this.

[WeirdOmen]

It would be nice if someone could add something about optics. I'm not expert enough to comment on it, but I do see that among the machines listed by Eric there are some who use Leds as excitation sources, others use lamps, and the number of emission filters (and multiplexing capabilities) vary accordingly.

[nonrad]

mchlmrmn - the well to well reproducibilty proves nothing? Really? Given that reproducible Ct's are not only the function of the block homogeneity but also the optics. I can't remember the name of the effect (parabolic or something?) but basically a lot of optics systems can't see the edges too well (longer light path) hence the use of ROX as a fluorescent normaliser. And perhaps a reason why a lot of people used to leave the outer wells of a plate empty with the older block-based instruments

But again, as you say, is the well-to-well variation an issue for the applications involved.

Regarding optics, lasers are expensive, lamps are cheaper. Some lamps have broader excitation that others (eg xenon) than the more typical tungsten. LEDs are cheaper still and have a long life-span - but single LED instruments have lower numbers of channels. . again, not a problem if you're not multiplexing.

[mchlbrmn]

mchlmrmn - the well to well reproducibilty proves nothing? Really? Given that reproducible Ct's are not only the function of the block homogeneity but also the optics. I can't remember the name of the effect (parabolic or something?) but basically a lot of optics systems can't see the edges too well (longer light path) hence the use of ROX as a fluorescent normaliser. And perhaps a reason why a lot of people used to leave the outer wells of a plate empty with the older block-based instruments

But again, as you say, is the well-to-well variation an issue for the applications involved.

Regarding optics, lasers are expensive, lamps are cheaper. Some lamps have broader excitation that others (eg xenon) than the more typical tungsten. LEDs are cheaper still and have a long life-span - but single LED instruments have lower numbers of channels. . again, not a problem if you're not multiplexing.

Oh yeah, I should have said "that test doesn't prove high PCR consistency", perhaps. I think the word you want is parallax? I think that was why the salesperson for the machine we got was talking about how long the light pathlength was in their machine, with mirrors, which would reduce parallax. That system has no ROX. I didn't know that that was the purpose of ROX, but it fits in now.

[nonrad]

So Eric - what are the applications in mind for the machine? And what are the abilities of the users?

[Wheygurt]

So Eric - what are the applications in mind for the machine? And what are the abilities of the users?

Well, it is difficult to answer your questions. This purchase is for different companies part of a business incubator. We (the committee) don't really known the skill of the people who gone run those assays because of companies discretion. We were ask to shop around for the more versatile engine and try also to extrapolate for the needs of other "unknown" companies that might join the incubator. So, applications could be from relative quantification, to genotyping and why not, some FRET-PCR on top. My feeling is that this machine will be use mostly for relative and absolute quantification. May be a company working on a specific plant or else where no specific primers are available yet, will need a machine that will help them set up their PCR reaction for diagnostic or quantitative purposes. I hope it answered your question.

Thanks.

Eric

[Suzanne]

Hi All,
OK- I have summarized all the answers so far. Is there anything else?
I noticed no one mentioned cost. There can be a wide range of costs for an instrument.
The #1 answer is run time- everyone wants a faster run time so they can get more work done per day.
OK- I'd like to make it a top 10 list so if you have 4 more criteria to add, post it now.
Suzanne


1. Run time: The most common request among everyone was fast run times so more work can be done in a day. It could be because the instrument is shared or because you have big projects and want high throughput.

2. Multiplexing capability. Many commented that the ability to multiplex is a plus point. You may not be doing it now, but its good to have the option in the future. Plus if it’s a shared instrument, the machine will need to have the flexibility to work in everyone’s project. To this end, asking the manufacturer about the type of optics becomes important. Lasers are expensive, lamps are cheaper. Some lamps have broader excitation than others (eg xenon) than the more typical tungsten. LEDs are cheaper still and have a long life-span - but single LED instruments have lower numbers of channels.

3. Flexibility of the plate format. You may have different projects / groups using the same unit. Some may have a 96 or 384 plate or even 0.2 ml tubes. A system that can use different plates will be a plus.

4. Ease of use. What is the current expertise of the people involved in real-time PCR? If you have a lot of new people, they will be calling technical support frequently. How is the company’s support? If it’s a shared instrument, will there be a designated expert to oversee the operation and help people internally?

5. Well to well variability. Some companies claim their machine is more accurate than rivals. When testing the instrument in house, try out some of your most difficult primer sets and make sure to use some of the outside and corner wells. A 0.5 degree well to well temp variation could have a major effect on PCR efficiency.

6. Warranty: most come with one year, some with two years. It is especially important for those systems have lasers to ask about the warranty options and figure that information into the cost.

[Suzanne]

Hi All,
Thanks for sharing your thoughts.

Here is the blog:

http://bitesizebio.com/2008/01/15/buyin ... cr-cycler/

Best,
Suzanne