Universal probe library probes.... - (Jul/23/2007 )

Hi all

I have recently started doing some real time PCR and was suggested to use Universal probes (ROCHE), and I have now got my probes and primers to start the assay.

one que though....does anyone know which quencher dye is used in these probes, I need to know this to start the assay on my machine!!


anyone working with these probes??????? are they good??


Rits

"They are labeled 5'-terminal with fluorescein (FAM) and 3'- proximal with a dark quencher dye."


https://www.roche-applied-science.com/sis/r...description.jsp

well, what machine do you use and what choices of quencher dyes can you adjust?

in some machines you just select between fluorescent (TAMRA) or non-fluorescent quencher (e.g. ABI) . in others there is simply no quencher adjustment (e.g. Bio-Rad).

you mean prism 7700? then just uncheck the quencher box, or select "none" instead of "TAMRA" or both

We got recently a Mouse set of UPL probes, since we're going to do a lots of different gene expression assays.

So far it works OK, I only have come comments to the Probe Finder application, it's not very easy to design differential assays or assays on specific part of a transcript and sometimes, when the RNA is small it doesn't show many primer options (and we even couldn't find any intron-spanning assay for one gene

But all the primers designed by PF are working good and assays are specific, however, efficiency and fluorescent level of different probes varies quite a lot (even when primer efficiencies tested on SYBR looked similar) and I don't know the reason.

I suppose it's good, when you need to design assays guick and don't want to be bothered much, but if you want something special (like differentiate between mouse and human transcript of the same gene) it's very difficult to push it through the application. Some people though design primers themselves and don't even use PF, didn't try.

It would be nice to exchange experiences with UPL further.

We got recently a Mouse set of UPL probes, since we're going to do a lots of different gene expression assays.

So far it works OK, I only have come comments to the Probe Finder application, it's not very easy to design differential assays or assays on specific part of a transcript and sometimes, when the RNA is small it doesn't show many primer options (and we even couldn't find any intron-spanning assay for one gene

But all the primers designed by PF are working good and assays are specific, however, efficiency and fluorescent level of different probes varies quite a lot (even when primer efficiencies tested on SYBR looked similar) and I don't know the reason.

I suppose it's good, when you need to design assays guick and don't want to be bothered much, but if you want something special (like differentiate between mouse and human transcript of the same gene) it's very difficult to push it through the application. Some people though design primers themselves and don't even use PF, didn't try.

It would be nice to exchange experiences with UPL further.

Hi Rits and everybody,

I also use the UPL assays for human gene expression and I really like it (especially the flexibility and fast assay design). You just need one set of probes and order your primers. Much cheaper than buying 1000s of probes or even premixed assays which I used before.
I know the issue with different fluorescent amplitudes of the assays, but this doesn't affect the result (Ct or Cp value which are determined at the "bottom").
Although it is not always possible to design your assay around 1 specifc base, you at least know where your primer and probes bind to your template which you do not know with ABI assays (only large pack sizes). For gene expression analysis this doesn't matter anyway.

The ProbeFinder SW has really improved over the past 2 years and I love the possibilty to do now Dual-Color Assays with the reference assays which sometimes is really great for gene expression analysis...

Troubleshooting hints:
-Sometimes it may be advantageous to increase the primer / probe concentrations to 500nM (each primer) and 250nM (probe).
-Use the Roche Transcriptor Kit for the RT-step. It is sometimes more sensitve than SSIII AND cheaper and forget the AB RT kits (insensitive and user-unfriendly) .
-The LC480 is more sensitive in detection therefor it works a bit better than AB instruments (especially the older generation of the 7x00s). However you can improve your assays with their FastStart Universal Probe Master (Rox) when using AB or Bio-Rad instruments (and others).


Would also appreciate to discuss more about UPL assays....

you mean prism 7700? then just uncheck the quencher box, or select "none" instead of "TAMRA" or both