Two things:

1. You have apparently a problem with the PCR per se, since you cannot detect the 2nd amplicon in the agarose gel. Most likely the PCRs interfere with each other. As long as this problem persists you cannot use these PCR together for your qPCR.

2. with SYBR green it is impossible to multiplex (as already said by Thomas it just binds to double stranded DNA without any specificity). In many cases, however, the multiplexing is not really necessary. The setting up of a Taqman or scorpion assays require some time and you have to think whether this is really woth doing it (unless there are probes published for your template that were shown to work nicely)

Good luck with your experiments

Volker

It is most likely that your primers for the 2 assays are interacting when the assay is run in a duplex format. If standard optimisation experiments do not work (increasing MgCl2, changing primer concentrations etc), try the addition of DMSO or Q solution (Qiagen - contains DMSO). Both reduce the interaction between oligos in PCR reactions and help remove secondary structure in the target.

A more obvious problem might be the annealing temperature used in the duplex PCR assay. Are you using the same annealing temperature for the duplex as for both of the simplex assays? I am sure you haven't made this simple mistake, but better to mention, just in case!

SYBR chemistry is not really ment to multiplex because the dye intercelates into any double stranded DNA complex. Try Taqman chemistry to multiplex.

Two things:

1. You have apparently a problem with the PCR per se, since you cannot detect the 2nd amplicon in the agarose gel. Most likely the PCRs interfere with each other. As long as this problem persists you cannot use these PCR together for your qPCR.

2. with SYBR green it is impossible to multiplex (as already said by Thomas it just binds to double stranded DNA without any specificity). In many cases, however, the multiplexing is not really necessary. The setting up of a Taqman or scorpion assays require some time and you have to think whether this is really woth doing it (unless there are probes published for your template that were shown to work nicely)

Good luck with your experiments

Volker

All of our research literature references for the use of "SYBR Green" have indicated the use of this DNA-binding flourophore for single transgene analysis. It is inexpensive and effective for that application; however, for multiplexilng it is suggested that you try Taqman chemistry from Applied Biosys. or some other reputable vendor. All the best!

I do think using such a chemical isn't a good idea indeed it does not concider unspecific ... For Qrt PCR, even if it is more expensive, I strongly advise you to use a reference gene with one label and your favorite gene with another....

It can save your experiment

1) I would try duplexing the 213 reaction with some other primer pair (not your 123), to see if it works in duplexing in general. If you are not strictly limited in your resources, and if the above mentioned experiment works, and your goal is to quantify relative amounts of 123 and 213 products, you may be able to find a primers-target combination, which works well when duplexed with BOTH of your reactions and run them in parallel. This way you will have an internal normalization (similar to the way 18S, or GAPDH, or other housekeepers are used in TaqMan).

2) I would also try designing new primers (for your targets, if this is possible) and testing different combinations for their efficacy in duplex qPCR (this may help, if, as Justin pointed out, annealing temperatures are different).

Good luck with your experiments

Vera

I agree with Justin that this is a typical interferences between two sets of primer sets. Basically, these two primers set are not compatible. Additionally, SYBR-green is not generally used for multiplexing with only one exception for qualitative detection providing the two target sequence are different enough to have different melting temperatures that can be differentiated using the melting curve analysis.

In your case, I think you need to redesign primers if you really want to do duplex q-PCR, which in most situations you don't have to. And depending your goal for the q-PCR, choose proper chemistry (in most cases, TaqMan is the best bet). Good luck!

First, standardize your end point PCR for separately by each fragment, next standardize duplex end point PCR and make sure you see the two amplicons on agarose gel.

SYBR chemistry is not really ment to multiplex because the dye intercelates into any double stranded DNA complex. Try Taqman chemistry to duplex o triplex if you use any internal control.

I think it is a valid alternative to TaqMan and it will be cheaper at the end per reaction than multiplex TaqMan. The amount of work for the optimization is also similar to what you have to do to make the 4 primers and the additional two probes work perfect (many expensive probe trial). Richard Cawthon suggests 10C difference between the amplicons Tm. But I agree that you should test the primers in a regular multiplex PCR and be able to produce the two amplicons, as a prerequisite. If there is copy number diff. (mRNA abundace) between the target genes, try to lower the primer conc for the multicopy or increase the other. Good luck!

SYBR green can be used in multiplex PCR When you design primer in such a way that you are getting different Tm then you can differentiate between them based on difference in melting temperature which could be specific.

Agree. You can differentiate the two product as one would melt away when you detect fluorescence from the second product.

SYBR green can be used in a multiplex PCR indeed, if the Tm and peaks of the different amplicons are separated enough, as mentionned above.

I will suggest to optimize your duplex PCR by trying different combinations of the primers concentrations, from 300nM to 900nM as in a matrice.

For example: 300/300 for primer A (Forward/Reverse), with 300/300 or 600/600, or 900/900, for primer B, and to do the same for all the different concentrations' combinations (including different concentrations between forward and reverse primers, like: 300/600, 300/900, 600/300, 600/900, 900/300).

PCR, in a singleplex or multiplex format, don't need to have the same concentrations for each of the primers in a pair.

This process is a bit time consuming, but it's worth it!

The second amplicon should be amplified by one (or several) of the primers' concentrations combinations. Select the combination where you get the best signal for your primers (lower primers concentration for the highest peaks / flurorescence).

If you still don't get an amplification from both primers, then your primers are definitively not compatible in a multiplex setting.

Hope this helps.

Forget SYBR.

Do you use one annealing temperature in order to get the two amplicons? Perhaps you should use two separate annealing temperature for each target amplicon and make the reaction go like this: x seconds 94 degrees for denaturation y seconds annealing temperature of 1st amplicon (the higher annealing temperature say 75deg) z seconds the annealing temperature of the 2nd amplicon (say 70deg) and v seconds for elongation. In this way you may give a chance to the primers of both amplicons to anneal.

For duplexing we always use TaqMan master mix (either fast or standard) with specific TaqMan probe/primers. You can not use Sybr green chemistry (as has been mentioned above) to run multiplex qPCR. Also remeber to have each of your primer/probes fitted with different 5' dye (normally one can be FAm and the other VIC). You can decide on your quencher (either NFQ or Tamra) depending on the design of your experiment. Hope it helped.

Quantitative analysis cannot be performed in multiplex with SYBR Green, but qualitative analysis in order to see if both amplicons have been amplified can be done, if the amplicons ha ve different Tm in thedissociation curve analysis.

I agree with previous posts that it is a problem of multiplexing. Often amplicons that work well in single pcr, may have problems when multiplexing. You can check if the two pairs of primers can work together with these software:

http://bioinfo.ut.ee/multiplx/

http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/

Additionally, you can try different ratios of primers but I would use lower primer concentrations: 100- 200nM for the 123bp fragment and 400-1000nM for the 213bp fragment. You should also use longer annealing times (90 sec - 4 min). The Mg concentration in muiltplex PCRs is usually higher (3-4.5 mM) than normal PCRs (1.25-1.75 mM), thus adding Mg is logical, but I don't know which is the Mg concentration in the SYBR-Green Mastermix. Finally you could try to use lower annealing temperatures.

Good luck

As mention, sybr is not ideal for multiplexing, choose another multiplexing dye

I would not advise multiplexing with the SYBR Green as you will be having problems with discerning the fluorescent signals of each respective primer pairs especially if they are targeting very close targets. Alternatively more robust chemistries you could try include TaqMan and Molecular Beacons relying mainly on dual labelled probes with two distinct fluoresecent dyes. All the best.

Good semi quantitatve data could be obtained with duplex PCR on gel or BioAnalyzer. I don't know if it's your purpose... In that case monitoring the amplification with Syber Green is a good thing. You can check the melting curve and stop the amplification before plateau for a good downstream quantification. I get very good results with the QuantiTect Multiplex PCR Kit from Qiagen (204541) in wich I added Eva Green instead of Syber Green.

Hi all,

Thank you very much for your valuable suggestions. I successfully standardized my duplex PCR using Power SYBR green master mix only. Special thanks to Claire Perrin & Seweryn Bialasiewicz for detailed explanation.

Great. I'm happy to hear that.

Can't we use sybr green for relative quantification with multiple genes? How far it is reliable?

Hi Siva! Can you briefly describe how did you suceed to duplex PCR with SYBR green, please? It uwould be useful to others too.

hi all,

Here is how I did my experiment. Real time duplex PCR was standardized by empirically varying the primer concentrations of gene A and B, alternatively. Firstly, Gene A primers are kept constant and gene B primers are varied and vice versa. Finally, i could get optimal amplification of both genes at 800nM (A) and 500nM (B).

Hi Siva,

I am trying to multiplex three primer pairs for a single gene to get three different products of approximately 180bp each. I used Powerup SYBR Green mastermix. the singleplex showed sharp single peaks. However, when I tried multiplexing it, one product dominated all others and there was only single product peak plus primer dimers. The efficiency of all the primers looks pretty good in singleplex. in addition to trying different primer concentrations, do you recommend anything else?

I appreciate your help.

If the Tm of your individual peaks differ by at least 3, then you can modify the melting stage to see difference in your peaks. I suggest you first amplify all three products separately, gel purify and mix at equimolar ratio, add SYBR green mastermix and perform only melt curve analysis by changing the ramping times. At a specific condition, you can see three individual peaks. However, I had done multiplexing with only two genes. Never with three genes.