There is no need to use ROX, but if you don't use it, you should tell the software *not* to normalize against ROX. Have you removed the check mark at "use ROX as internal standard"?

The mastermixes do contain ROX and it's best to just leave the normalisation on.

I'd suggest running the qpcr products in a gel to see if you have a product. Then you can at least eliminate the possibility that the amplification didn't work.

HI Jesica

I also agree with suggestions given by Jochem and Fiona.....first of all you make sure you GOI interest is amplifying in PCR or not...and as far as ROX is concerned....follow the manual of the Kit your are using...many kits provide the ROX as a separate vial..so it is up to you to use it or not and that is decided by the qPCR machine you are using...many of them use internal calibration methods and do not require ROX to be added to the PCR tubes..

bye

Roche sells several master mixes for RT, some with and others without ROX, therefore its difficult to answer your question without knowing the name :-)

NO the Roche don't need ROX for qPCR. The ROX is for normalization the reactions.

Hello Jesica, I agree with the comments posted by other investigators regarding the need for ROX. However, one important point here is the manufacturer of the qPCR instrument is ABI and the manufacturer of the master mix is Roche. Roche makes many different master-mixes for qPCR, each with a unique chemistry designed for specific applications and intruments. You need to make sure the mix you are using is compatible with ABI platforms. Also, you need to make sure of the type of PCR block that is in your instrument, is it for normal 9600 PCR cycling, or is it set for rapid cycling? I would suggest that before running more experiments you check buffer/instrument compatibility and the type of cycling (normal or rapid) your instrument uses. Another point to check is if your master mix is good, do you have a positive control sample for something unrelated, if so run a reaction to see if it works. It is surprising that you do not have signal with b-actin, expression levels of this gene are very high. Check the quality of your RNA, if it is degraded you will not have good results. Anyway, keep me posted and I can help you troubleshoot as you move through these suggestions. Good luck.

I have tried using Roche master mix with ABI using different concentrations and what not... It does not work. As suggested above, there is a compatibility issue.

@ Roberto and Vartika,

I know well the strategic advertisings of these companies trying to sell own assays tailored to their instruments. I was never able to understand what a reason could be that an assay should work with one instrument but not with another.

The instruments heat and cool the reactions. They irridate the reactions with light and they measure emmitted light. Given that the acceptors/fluorophores used fit to the wavelengths of the excitation and the filters fit to the emmission, how can it depend on the instrument whether or not a reaction (given its components and concentrations like Mg++, PCR enhancers, polymerases, dNTPs,...) will work?

Would be great if you could help me there.

Hello Jochen, I cannot give you an exact answer all I can offer is speculation. Originally Roche developed its buffer composition for real-time PCR optimized for the light-cycler, while ABI pursued optimized its PCR chemistry based on the 9600 platform and use of Taqman probes. In theory, the chemistries used should be cross-compatible, however, as many of us know, a PCR reaction that works well on one instrument may require optimization if run on an instrument from a different vendor. Another distinction is the source of the polymerase, different vendors, different enzymes, different PCR results for assays run on the same instrument. Roche does sell a master mix that is optimized for the ABI instruments and that is compatible with Taqman probes. Yet, this same chemistry does not work well on the LC480 instrument manufactured by Roche. I have had experience working with these different master mixes and tested them on the Roche LC480 and ABI 7000, 7500, and 7900. I have found that what works with one instrument Roche or ABI does not perform well on the other. I hope this helps.

I have to mention that I have the correct bands I saw them in a agarose gel , the problem is that the Applied biosystem can read the signal with my reagent!!!! Now I`m going to perfom some experiments to elucidate if it is a problem of my lote or a problem of the compability. So fas as I know I will comment here my results. The only that I knew it is that another person works with the same equipment and another master mix and this works well! Well, I have to go on with finding the resolution of this! my experiments are stopped beacause of that! :(

Hi Jesica, what is the detection based on Taqman probe?

If you are using LC-Green it will not work on the ABI system. The emission spectrum is not detectable by the ABI instruments.

Thank you Robert. I used different SYBR-Green mastermixes, fom ABI, Roche, and third-party vendors on both systems, LC and SDS. All mixes I used worked on all instruments. In fact, I got the "best" amplification curves (lowest SNR, most points in log-linear phase) when using a Roche mix on the SDS. But, evidentially, you made some different observations. I never tried mastermixes with sequence specific probes. When I was working with such probes the commercial mixes were suboptimal and I used to make my own mixes (that performed much better, for sure ;) ).

so, again! I bought this reagent: Faststart SYBR Green Master mix, apparently without ROX. So, it is not convenient for ABI 7500!? Is that what you are trying to say!? The point here is that a colegue use this combination ABI 7500/this master mix without problems so I don`t understand what is the difference!

and other thing: I didnt understand, independently that the mix have or no ROX, the equipment requiere it always!? or have a mode where you put without normalization and work well!? If I put ROX in this master mix, it will work!?

regarding to de chemistry: I don' t use taqman probe, I work with SYBR Green chemistry. Another thing that I dont understand is those that you mentioned before about both systems, LC and SDS... could you explain me!?

Hi Jesica,

apparently, you're facing more or less handling problems, if the same assay works with a colleague of yours. Maybe you could ask your colleague to prepare a qPCR plate with your samples and kit to be sure, that it's not bad qualitiy of your samples and kit components. Other than that I can only give you general advice like: be careful when sealing the plate not to make any scratches on the transparent surface as they will influence the transmission of fluorescence light, spin the plate carefully after having sealed the plate to make sure all components are able to take part in PCR reaction.

For the ROX-story, I know people who like to use ROX, because it slightly amplifies the fluorescent signal and also normalize the signal (see comments above), in fact I don't use ROX when working with SyberGreen and I never had any bad experiences with missing signals (also using ABI cycler). Some people also recommend not to use the outer line of wells in a 96 well plate, because the angle of the laser differs to the one when focussing on the inner wells, resulting in lesser fluorescence signal being detected (which I only observed once, but tend to ignore it because it usually works fine for me using all wells...). What also is important to consider is actually the type of plates being used in your qPCR-reader. But you can easily identify the recommended plates in the manual of your device (look here: http://iris.fishersci.ca/LitRepo.nsf/0/B8A59DD204B5515D852575F5006B45F5/$file/ABgene%2096-Well%20PCR%20Plates%20%28Fisher%20Sci%20Version%29%20low-res.pdf) and last comment: I always use white-wall plates instead of transparent ones for better reflection of fluorescence light... good luck and keep us updated about the progress of solving the problem ;)

Jesica,

To the best of my knowledge and experience, Faststart SYBR Green Master mix (without ROX) works on the ABI7500. This is also confirmed by the fact that your collegue is successful in using it. You should find out what the differences are. I suggest that you take one ore some of his cDNA samples and he takes some of your cDNA samples to see if already the samples are problematic. See also the comment of Martin above.

The equipment doen't recognize and doesn't depend on the absence or precence of ROX. If Rox is present, its fluorescence can be measured and the fluorescence from the reporter probe (SYBR-Green for instance) can be normalized to the ROX signal. This normalization will cancell out (more or less!) some fluctuations in fluorecence intensity that are independent of the fluorophore (e.g. induced by temperature differences, pH differences, volume differences, noise of the irradiation and detection system). On the one hand such sources of "noise" may be eliminated (or lowered) but on the other hand this may introduce "bias" (if the influences are *not* similar on ROX and SYBR). Unfortunately, little (independent) research is done on this question, so it's more or less up to you taste whether or not you like this internal normalization. Clearly, *doing* this normaliziation *without* having ROX in the mix will increase the noise, because the reporter signal will be divided by a complete noise signal that it close to zero. This may result in very, very noisy amplification curves (depending on the baseline fluorescence intensity at the wavelength where ROX is measured). All software I know allow to turn this normalization on and off. It's just about cklicking a checkbox somewhere.

LC = LightCycler, the Roche Instruments. Roch favours the use of hybridization probes (because they hold the patents on this).

SDS = Sequence Detection System, the official name of the instruments sold by PerkinElmer, then ABI, Applied Biosystems or whatever the company name is changed to. ABI favours hydrolysis probes, that are often called "TaqMan" probes (jargon) bequase a propery of the Taq polymerase is used to chop down the probes, like the good old PacMan was eating up the yellow dots on the screen. Therefore people started to call the instrument the "TaqMan machine", what finally turned out to be genius strike of the company making users believe that this instrument really requires the "TaqMan" chemistry (what is wrong, though!).

thanks all for yours answers! I wrote to the enzyme supplier and to the Applied biosystem support team and I had two answers... the first ones said to me, maybe because they want I to buy a new master mix, that with my equipment an enzyme with ROX is better... then the technician of Applied biosystem said that my problem was very easy to resolve... I had to check out the item of ROX at the beggining of the set up. And was easy, I did that and reanalyzed my samples and then all the data appeared in the results window!!! :) So It was a little detail that obviously it is very important!

So my very first answer (the second in this thread, 8 days ago) was absolutely correct and would have right away solved the issue ;)

I´am trying a new kit, and they sells you the ROX separately. Do you know wich final concentration of the dye you must add to the mix? My solution its 50x