Best chemistry for relative quantitation of gene expression - Give your opinion on the best chemistry according to your experience (Oct/03/2008 )
Hello everyone! I am a brand new member in BioForum. In fact, I found it looking for the probe finder software from Roche and for what I had read so far, the forum is great. I would really like your opinions on a tricky question: what is the best chemistry for relative quantitation of gene expression? And I mean not only from the theorical point of view, but also the practical aspects of it. For instance, I read in this forum that some companies take more time than others in handling your probe order. I have a little experience on the topic and I would like to share it with you. I work with bacteria (perhaps many of you do not) but at the end, gene expression is gene expression. I had meassure expression of 5 different genes using two different chemistries: TaqMan probes and SybrGreen. The only complain I have with TaqMan probes is that at least in my case, they take forever to synthesize my probes (Assay by Design system from ABI). So I decided to switch to SybrGreen and it worked fine, but the standarization took me sometime. Now I am planning to quantify the expression of other 5 genes. I have to start soon to desing primers and perhaps probes. When I looked at the variety of chemistries there are now, you wonder if you are making the right choise or if there is something else out there that you missed. For instance, I was very tempted to use the Lux primers, but someone told me that they just don{t work. So I did not even tried. Now I found that the Universal Probe Library can be used with any organism (at least that is what Roche claims) and that the probes are prevalidated...According to my experience in RT-PCR this sounds to good to be true. I am using a 7300 ABI. Any input is of great help!
AFAIK there is no absolutely universal solution to real-time PCR, it depends on what your familiar with, the money you have, the assays you need.
Many people are happy with SYBR for gene expression while others do a probe assays, because they believe they're more specific. I'd would agree with that, but probes are more expensive and more trouble to design, to be sure it would work.
I have a personal experience with the Universal probe library and a good one I would say. It's as it says, it covers any organism (two packs of probes), or you can order separate ones for Human and Mouse genomes (but of course most of the probes are the same, so we have a Mouse pack and can design assay for human gene without a problem). The principle of the UPL is different from classic Taqman probes, so everything is covered by only 165 small probes (for more info read the Roche UPL homepage).
When you purchase the probe pack, you only need the Probe finder software on Roche pages, it will design an assay for you, (in fact it's essential, you can hardly design assay by something else, since you don't know the probes sequences). You only order primers and the run it on predefined conditions, no optimization is required in about 95 % of cases (and the rest you just order another primers, you don't optimize, it's a waste of your time and material).
We have it for a year or so, and made more than 40 assays with it, it works wery well on normal sized genes, problem comes when you have a gene too short (like a globin gene) and too homologous to different genes (like a globin gene) or you want to amplify just-that-part-of-the-gene, but for a standard expression studies it is remarkably easy and cost efficient (compared with purchasing every probe for every assay). Really saves an awfull lot of time, I'd recomend it if you plan to do many gene expression assays through the years.
(and by the way, we sometimes use Probe Finder designed primers for a SYBR green only and didn't have big probles with it, so you can try that too, the primers are indeed designed (and tested in silico) to work with UPL probes, but they must work fine on their own too)
Thanks a lot for you input! And what about UPL delivery time? How long does Roche take to deliver the probes? Catichu
Many people are happy with SYBR for gene expression while others do a probe assays, because they believe they're more specific. I'd would agree with that, but probes are more expensive and more trouble to design, to be sure it would work.
I have a personal experience with the Universal probe library and a good one I would say. It's as it says, it covers any organism (two packs of probes), or you can order separate ones for Human and Mouse genomes (but of course most of the probes are the same, so we have a Mouse pack and can design assay for human gene without a problem). The principle of the UPL is different from classic Taqman probes, so everything is covered by only 165 small probes (for more info read the Roche UPL homepage).
When you purchase the probe pack, you only need the Probe finder software on Roche pages, it will design an assay for you, (in fact it's essential, you can hardly design assay by something else, since you don't know the probes sequences). You only order primers and the run it on predefined conditions, no optimization is required in about 95 % of cases (and the rest you just order another primers, you don't optimize, it's a waste of your time and material).
We have it for a year or so, and made more than 40 assays with it, it works wery well on normal sized genes, problem comes when you have a gene too short (like a globin gene) and too homologous to different genes (like a globin gene) or you want to amplify just-that-part-of-the-gene, but for a standard expression studies it is remarkably easy and cost efficient (compared with purchasing every probe for every assay). Really saves an awfull lot of time, I'd recomend it if you plan to do many gene expression assays through the years.
(and by the way, we sometimes use Probe Finder designed primers for a SYBR green only and didn't have big probles with it, so you can try that too, the primers are indeed designed (and tested in silico) to work with UPL probes, but they must work fine on their own too)
Delivery times here are about two-three weeks, but you should ask Roche representatives, they will have the answer relevant for your region.
I'll just add a few opinions
upl probes sound like a good idea
I have got probes from BioSearch Technologies and it's only taken a week or so
FAM taqman probes of usd 100 or something, you could check with them on prices and how long it would take.
I'm not in the USA and our exchange rate has plummeted so it might not be the best option for me anymore, but your situation might be different and if you're in USA then I would have thought it was a good option.
I've had a lot of experience with LUX primers. I was doing a duplex reaction using FAM and JOE LUX primers. The reactions were working beautifully on a RotorGene 3000 but when I tried the same reaction on other realtime machines things didn't look so good. I think some machines struggle with the small change in fluorescence that you get when the LUX primer becomes incorporated into PCR product.
If you are doing a simplex reaction then I don't know why you would use LUX primers instead of SYBR or SYTO9 or whatever.
Cheers
TK
Thanks a lot for your input! Things are becoming more clearer now! Catichu
upl probes sound like a good idea
I have got probes from BioSearch Technologies and it's only taken a week or so
FAM taqman probes of usd 100 or something, you could check with them on prices and how long it would take.
I'm not in the USA and our exchange rate has plummeted so it might not be the best option for me anymore, but your situation might be different and if you're in USA then I would have thought it was a good option.
I've had a lot of experience with LUX primers. I was doing a duplex reaction using FAM and JOE LUX primers. The reactions were working beautifully on a RotorGene 3000 but when I tried the same reaction on other realtime machines things didn't look so good. I think some machines struggle with the small change in fluorescence that you get when the LUX primer becomes incorporated into PCR product.
If you are doing a simplex reaction then I don't know why you would use LUX primers instead of SYBR or SYTO9 or whatever.
Cheers
TK