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Sybr Green vs Taqman -- a practical approach

qrt-pcr sybr green taqman

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#1 Zee

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Posted 28 June 2013 - 04:44 AM

My PI wants me to switch from using Taqman to Sybr green to look at expression of a non-endogenous gene. Mainly the switch is to safe money. I'm not convinced that this is the best idea as Taqman is supposed to be more accurate. Also, a friend of mine said that her old PI determined that Sybr was not that much cheaper. She said this was because of the large number of controls you have to run. Since I have never run a Sybr green experiment so am unfamiliar with the number of controls required, I was wondering if anyone has compared the costs between the 2 methods. How many controls are you supposed to run? I know when you get a new primer set you have to run a standard curve, but you don't have to do that every time, do you? Are there additional controls I am not thinking about?

If you have any arguments for either method please let me know so I can either agree that the switch is a good idea or approach my PI with arguments to not switch.

Thanks for your help!

#2 Curtis

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Posted 29 June 2013 - 07:31 PM

Don't worry, in fact many people prefer SYBR green if the target is not really that specific. There are some companies that produce already-optimised qPCR kits. I used Qiagen's line of Quantitect products that were optimized for the Rotorgene Real-Time PCR machine. Rotorgene is a product of the Australian company Corbett, and you must check the name of the machine you want to use as well. My kit was already optimized for that machine so I didn't run many optimization runs. Number of controls also depends on your experiment. Quantitect have a large archive of primers for many known genes in many different species that you can just log on to their website and chose the primer pair:

http://www.qiagen.co...ringinformation

and chose these PCR kits

http://www.qiagen.co...ringinformation

I am just a moderator here, and don't advertise for any company. I'm just saying that there are ways to speed up your research. I am not saying that you only buy from Qiagen, because that could be expensive for you too, considering your location and shipment cost to your country. The Primer Assay kit costs $85. You can check the cost of the PCR kit yourself. If your machine is not listed in their website you can order your own primers and use a cheaper SYBR green kit. I have seen many people use unknown products from unknown companies and yet get their qPCR to work. SYBR green is not that difficult.

#3 Trof

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Posted 29 June 2013 - 11:40 PM

SYBR can by less specific from the principle, but it also includes his own "internal control" the ability tu run melting curve and check the specifity.
Actually as probes based systems are supposed to be accurate, the is no way to tell if they don't detect two different products for example (you can only guess this, as your PCR efficiency will be lower than it should be). With SYBR all that's double stranded is detected and you can check it.

This means also on the other hand, that while truly specific probe based system can give you quite accurate results even if your primer amplify more products, because probe detects only the specific one, with SYBR you need to optimize your reaction so there are no nonspecific products or primer dimers. But you can check everytime if they are. This way you know you can't use your results.

I personaly first run every primer pair on SYBR to check specifity and dimers before I run them with probes.

The prize may also depend on the numbers of different assays you run, how many runs overall for each assay and how often you need new assays. Designing probes for every assay when you need 20 new a month just for measuring few samples, make the probe-by-design very expensive, on the other hand if you were just running three different assays in thousands for years and years (like in diagnostic lab) you can buy larger amounts and probes don't get waisted.

Another solution that we choosed, since we are the case that needs new and new assays all the time, is the UPL system, you buy a set of probes (the initial prize is quite high, around $5000) that very likely can fit any human (or mouse or so) transcript, and then you only order primers from now on. We have it for several years now and only buy new individual probes it's used up. Advantage is, if the first ordered system doesn't work or something, you can order different primers and use with different probe and in most cases it will work then.
Only problem we encoutered was with a very short transcripts and in cases we needed probe to be on exact exon-exon location and so, because the probes are not always available there.

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