There is no intrinsic reason that Taqman would be required over SYBR green. If you perform the problem quality control experiments for your SYBR qPCR, you can convincingly demonstrate specificity. Check the PCR efficiency, confirm that you amplify a single product using a denaturation curve, design your primers carefully such that they cannot amplify genomic targets, but instead only mature mRNAs (target exon boundaries if possible).

If you want to make absolutely certain that you achieve target specificity, you can always sequence your PCR product to prove that you amplified the intended seqeunce.

There is no intrinsic reason that Taqman would be required over SYBR green. If you perform the problem quality control experiments for your SYBR qPCR, you can convincingly demonstrate specificity. Check the PCR efficiency, confirm that you amplify a single product using a denaturation curve, design your primers carefully such that they cannot amplify genomic targets, but instead only mature mRNAs (target exon boundaries if possible).

If you want to make absolutely certain that you achieve target specificity, you can always sequence your PCR product to prove that you amplified the intended seqeunce.

Thank you! This is going to be my first qPCR as an undergrad and your answer helped a lot!

I totally agree with Dr. Cohen's comment. Both methods should work appropriately.

If you are concerned about publishability you may want to take a look at the MIQE guidelines: http://www.clinchem.org/content/55/4/611.full.pdf

And I agree with the responses above, both methods should work and also SYBR is straightforward and cheaper than Taqman.

SYBR Green assay I think is better for your experiments. Thank you

Primers and probes are more specific

Hi,

Both are easy to work with.

Taqman probes are standardized and you get to choose from there big collection or you can custom order. Since they save the hassle of standardization for efficiency except the standard curve generation for various conc. you do everything easily. Since its a fluorescent dye chemistry its more specific. It just doesn't bind to any double stranded entity but is only specific to the region of interest. hence the cost can be more. Taqman is surely more sensitive. A good quality RNA is always recommended for a better amplification efficiency of your cDNA.

SYBR is good if you doing bulk work, saves money, but you need to have your own primer sets to get them standardized for their specificity and avoid gDNA contamination. Its a little lengthy procedure but in any case an appropriate endogenous control always helps in both the methods.

KP

You can choose from wide range of Taqman probes, which are highly specific and sensitive with good fluorescent dye chemistry. since for cDNA work good quality RNA is essential and this is first experiment for you, go for Taqman and then after as per skill and requirement, you can go for either Taqman or SYBR green.

The only thing I would like to add. If possible use EvaGreen instead of SYBR Green. I think most people will agree that EvaGreen is better regarding signal to noise ratio, price and most important melting curve analysis.

http://www.biotium.com/product/product_info/newproduct/evagreen.asp

Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007)

I don't think Sybr green or Taqman reagents can affect the results in any way, both are equally effective. PCR efficiency can be good or bad, with any of these. Sybr Green is less expensive. Taqman is customized and sure-shot for initial assays. But once standardized, any of these will work with equal efficiency. Though have been a dedicated user of Taqman, must admit it is expensive.

I agree with Sarbani that Taqman usually works on the first shot, but the pre-designed assays are not infallible. I've only run into one or two Taqman assays that failed (targets could be detected by SYBR green or other methods), but it is important to note that Taqman can yield false negatives (false positives are not a concern, though).

I only use SYBR green for qPCR assays-it is a straight forward technique that is more flexible in terms of using it with a wide range of primers. All points made above regarding SYBR validation are excellent IE checking efficiency, checking product on gel, sequencing products-follow these and your results will stand strong for publication.

In my opinion it is not so obvious which one (SYBR or TaqMan) is better. Maybe due to chemistry TaqMan is more sensitive but still prone to nonspecific amplification. In this case you will not see and quantify the nonspecific amplification but in case it happens it influences reaction efficiency. So for TaqMan one should perform SYBR Green reaction (same primers, no probe) to check for product homogeneity to make sure polymerase is engaged in amplification of specific product only.

Really Taqman probes can be good to asses the expression level of you shRNA as it has good sensitivity for relatively short and structured targets. SYBR should be just fine for mRNA expression.

If your PCR product is small (less than 100 bp) in my view, it is better to not use SyBr Green. If you are worried about some unspecific annealing of your primers and therefore some unspecific signal then using a fluorescent probe will add specificity.

The only issue with SYBR is that it quite possible that melting point analysis will be unable to distinguish your target amplimer from a misprimer amplimer. But at least one sequencing would be required for both SYBR and TAQMAN methods anyway.

Taqman is an extremely robust method in terms of specificity, but that isn't always required anyway and Taqman is much more expensive. Those dye labelled primers end up costing you a fortune....

Unfortunately primer manufacturers sometimes play the bait and switch game once they think you have locked into a assay with their fluorescent probes. They will initially offer you a good price per batch, but then the primer yield will mysteriously drop 2-10 fold so the price of the primer increases 2-10 fold.

Taqman is a very good method though, that gives very predictable results.When you use a robot and the right master mix you can do 2-3 primer/probes per reaction including an internal control.

I think SYBR green assay would be more straight forward and convincing.

Use Roche UPL System. Its is straight forward to design and higher specificity than SYBR green

I have used quite a few Taqman assays from ABI. They've worked, but be aware that, in spite of what ABI says, only about half of them had close to 100% efficiency. I got all the failures to 100% by doubling the [Mg], but it's absolutely critical to check the efficiency of all assays. (For SYBR assays, mostly what I do, I like SYBR GreenER from Invitrogen. I suspect it's similar to EvaGreen--see above--in having a better S/N than straight SYBR Green.)

@Corine, could you please expand on your answer? My former lab routinely used SYBR with products smaller than 100 bp. I'm curious why you think this was not a good idea.

Double-stranded DNA binding dyes are cheaper and more versatile, in that you can use them with any primer set. In addition, you can perform a melting curve with dye-based qPCR, which you can't with Taqman assay. This gives you some additional support with each sample that what you're quantifying is the correct product.

To get the same level of certainty with Taqman, you'd have to run product on a gel to verify its identity (which will have lower sensitivity for picking up incorrect products like primer-dimer or genomic DNA contamination), or sequence your products (which would also only give you the identity of your majority component).

As has been suggested elsewhere in the comments, newer dsDNA binding dyes are brighter than SYBR. Promega makes BRYT Green dye which outperforms SYBR.

Most of my SYBR (GreenER) assays DO have amplicons less than 100 bp and I'm not aware of a problem. I assume specificity is a function of primer size, not amplicon size.

I'd have to agree with Deborah and William. The smaller your amplicon the more pronounced any differences will be, and consequently the higher your resolution. When doing a high resolution melt you're often told to keep it between 75-150 bp, and the smaller the better.

Also, if you're going with a melt I would suggest a saturating dye like EvaGreen (I think someone else mentioned it). A non-saturating dye like SYBR green can have the released dyes reincorporate into downstream gaps and throw off your reading a little bit. Not really a big deal if your strands are really different, though.

Price wise sybrGreen is better, you design your own primers and you sequence the product (better if size between 150 and 200 for sequencing, but you can use shorter too you dont need perfect sequence). Taqman is harder to design so to be safe researcher normally use the one design by the company, it is more expensive and you still need to do some amount of checking.

Reviewers are unlikely to make a fuss for the QPCR method, unless they are no very knowledgeable, but they could demand a western blot or some functional assay showing that the protein is down as knocking down mRNA is not necessarly enough to really affet protein level enough to have an effect, but that is a problem that is really system dependent. Remember you cannot cover for everthing that reviewer might ask so the first step is to convince your-self and your team-mates.

Wow... this site is AMAZING. I really wished I knew this place in my freshman year. But anyways, my amplicon is around 600bp. My plan is to check if the primers are specific by performing PCR and sequencing as the sequencing service from a company called Operon only cost me about 7 dollars. Right now I have 3 pairs of primers and the sequencing part would not start until I find out that the primers are working properly. So I would not be worried much about non-specific amplification. I guess I watched a lot of commercial promotion clips about Taqman probe that I started to think "is this one more preferable nowadays?" I realized that SYBR Green would save me a lot as I don't have to design probes. By the way, I am working with A. aegypti mosquito so I am not sure if there are any standardized probes. Thanks everyone!!!

In general, qPCR cycle times are designed to assay amplicons in the 70-250bp range. 600bp is a bit large, and it is may be unnecessary to target such a large sequence.

For qPCR assay 600 bp is too large. Better to design primers to obtain amplicon size within the range of suitable amplicon size for qPCR assay. If you have specific primers, both assays would be working fine. But as many others mentioned theSYBR Green is cheaper than TaqMan assay.

SYBR Green chemistry is versatile and cheaper. Follow the MIQE guidelines to verify your results when using SYBR Green. I prefer SYBR Green over Taqman because of it is cost effective and verifiable. One time, I had to opt for Taqman since it was difficult to design many primer sets and verify them for a gene with many pseudogenes. However, when I asked the company about the guarantee of the specificity of the Taqman probes that I ordered, no explicit guarantee was offered. So, beware!

i agree with Brat and Daniel

SYBR is enough and it is also simple and cheap.

I would recommend to follow the MIQE guidelines available on http://www.gene-quantification.com/

Taqman is better for em although both work ok!

Good luck!

You should always stick with the Master mix that the company recommends for their Taqman primers. For SYBR Green, you can choose that gives optimal results. In our experience, Takara-Clontech Premix Ex Taq DNA polymerase (Perfect Real Time) kit works wonderfully when we compared to Master mixes from three major vendors.

i agree with Daniel.

SYBR green is less expensive and there is no clear advantage to use Taqman over SYBR green for straight forward gene expression studies

Just be carefull when designing the primers, and then use SybrGreen as it is much cheaper. I have used extensively RNAi and do not require Taqman for kd validation. Just characterise your primers, check dissociation curves and should be fine.

I want to stress that your validate your primers following MIQE guidelines. Not all primers that are published are valid. If you blast these at the NCBI website, some of them are not even found in the genome. and in my experience most cannot be validated. They don't amplify at all and even if they do they fail melt curve analysis and some form primer-dimmers as revealed by gel analysis. Be ware! If you have validated primers, SYBR Green is cheaper and works fine. No need for expensive Taqma probes. Even if you use Taqman probes, you can verify them on a gel for the absence of primer-dimers.

Both work fine, although, working with taqman probes is less time consuming.

As some other people have already said, I think SYBR green can be absolutely fine if you design your primers carefully and have adequate QC. I used SYBR green as it is more economical. I combine it with predesigned and optimised primer assays from Qiagen. I found that these were excellent as they all anneal at the same temperature, allowing multiple primers to be run on each plate. I have never had a problem with primer dimer with these so no need to add in extra melt steps and they always appear to work first time with similar efficiencies.

I forgot to add earlier that I like to be able to see the melting curves for the products, something that can be done with SYBR but not with Taqman. Melts help visualize multiple products from a reaction, and primer-dimers. And, since Tm generally (not always) increases with product length, it can give some indication of whether the product is likely to be the desired one. (For further confirmation, I occasionally run gels on products from both kinds of reaction.)

Both work fine. However you will need to design and optimize taqman probes.

http://www.ncbi.nlm.nih.gov/pubmed/23176309

Srividya Subramanian, if you use Sybr Green, then analyzing your products on gel to check for presence of primer dimers is unnecessary step. You can visualize dimers by melting curves.

I agree with Denial Cohen If you like to use SYBR green for qPCR, I think following melting curve is essential. You can replace SYBR green with less expensive 20x EvaGreen in qPCR followed by melting curve.

I would like to add to the answer of Chandramohan Ishwad that the main advantage of EvaGreen is not the price but the fact that it does not inhibit PCR like SybrGreen.

I agree with most of the people that SYBR green will work fine just make sure primers are fine and as one concern with BLAST using NCBI site is raised, I think preferably use the advanced updated genome browser for you species and then u are sure about your transcript and for dimers u can check with various online tools as well. At end, you can check melt curve will tell everything. I find this one better-

http://cgi-www.daimi.au.dk/cgi-chili/primique/front.py

We have used both types of assay. SYBR green is cheaper than Taqman, and I have found no reason not to use SYBR green for routine qRT-PCR.. You can readily design your own primers, which may save money.

Keeping an eye on melting curves is esessential, but most real-.time PCR instruments will do this for you.

I usually prefer TaqMan because of the increased specificity with the use of a probe. But it's more expensive. If you have the budget, I would say go with TaqMan. That being said, I have seen many people use SYBR Green and get good results. Just be sure to check on the specificity of your amplification.

TaqMan is more expensive, but is way more specific than SybrGreen. If you choose Sybr is neccesary to run a melting curve and demostrate that your amplicon is the one you are looking for.

If you get budget, go for TaqMan, if not, you can work with Sybr, but remember make clear your amplicon is right.

SYBR Green is recommended here that too for publishing quality results. More vendors of molecular biology kits prefer this. Our lab uses 12.5 microlitre SYBR Green mix for a total reaction volume of 20 microlitre (including the 10 times diluted cDNA). We have already published. So you can go for it.

For difficult PCRs, for example genes with low expression (and in the RNAi, you want to get as low as possible), use Taqman. Looks expensive, but in reality saves more money than weeks devoted to optimization of Sybr Green. And it is very reliable.

Dye-based assays are perfect for many real-time qPCR applications. When you understand the true advantages and disadvantages of probe-based assays, you can reduce the cost while keeping the quality of your qPCR experiments high.

Novice qPCR users often argue that data generated with dye-based assays are susceptible to misinterpretation: the detection of nonspecific PCR products would influence the Cq values.

But the detection of nonspecific PCR products should not be cancelled out by adding a probe to the qPCR reaction. It is through careful experiment design and optimization that you obtain specific, efficient and sensitive amplification and detection of your target of interest. Primer concentrations, denaturation and annealing times and temperatures, and buffer composition are few of many critical parameters that contribute to a robust qPCR reaction. When sensitivity is not an aspect that you want to improve, optimizing these parameters will give you a suitable qPCR assay. And it does not need a probe. This is the strategy applied by Biogazelle when designing Bio-Rad's PrimePCR assays: the most comprehensive portfolio of validated Research Use Only dye-based qPCR assays for human, mouse and rat.

You can find additional arguments, and additional advice on when to use what detection technology at https://blog.qbaseplus.com/taqman-probe-or-sybr-green-dye

Hi! Does taqman probe technology give more sensitivity the SYBR green?. We have to work with low expression and little materials, which is the best technology?

Thanks,

Christian

Hi Christian, I have used TaqMan PreAmp Master Mix Kit for my real-time PCR experiments (Applied Biosystems) TaqMan PreAmp Master Mix Kit contains TaqMan PreAmp Master Mix and TaqMan Gene Expression Master Mix. These two reagents worked well for preamplification and quantitation of very low amount of mRNA of interest in my precious samples.

Hi!

Everyone has given useful answers.

I will add that if you have direct access to the qPCR machine I am in favor of Sybrgreen, because you can try two or three different assays for the same price.

I was so impressed by the claims of the designers of TaqMan assays; that they were validated and guaranteed accurate results, that i invested in their technology. However, some of the probes that I ordered gave scanty and imprecise results, while the Sybr Green assays that I used with either primers and probes that I found in published literature or designed myself gave consistent, good results that I was confident to publish. Hope this helps.

Taqman is more specific than Sybr Green.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988599/