slide assay with vista green. It's possible?

[Peter Ellis]

GysdeJongh wrote:
>
> Our microarray facility also changed a lot.My friend now runs the
> place. He is a good biologist but also a good manager . He cleared
> out the whole lab in just one day. Now there are labels "Affymetrix"
> everywhere . There are now 2 statistitians and 3 bioinformaticians.
> There is no printer. No body except  the very skilled technitians
> enters the lab ; you may handover yout RNA at the door . Compugen is
> doing something else , they nolonger sell the ologo library.
>
> By that time I wandered are we clumsy ? Can other people do this ? So
> I have done this already :

Ah right, it seems you and I were using different definitions of "home 
brew".  I guess I simply have the luxury of working in a University with a 
well-run and active array printing facility.  I certainly would *not* 
recommend for any small group to try and get the whole array fabrication 
process off the ground by themselves.

However there certainly is a niche for people to provide their own cDNA 
libraries, have the chips printed by an established facility, and then go on 
to do good publishable work with those chips - it's simply not the case that 
you have to buy into the Affy in-situ-synthesised-oligo market.

Quite apart from anything else, our trypanosome group would be waiting a 
*long* time for Affy to make a trypanosome chip!


>
> The surprise was indeed pleasant : we were not alone. We both know
> that the word "Affymetrix" could still be only in the methods section
> where this search does not look. I  _did_  look at all the papers I
> could find by the search above and other variations. I found two
> types : 1) home brew chips by a large microarray facility adressing a
> technical issue , 2) home brew chips by a large microarray facility
> working very closely together with a large biomedical group adressing
> an interesting biomedical issue. I found zero articles by a small
> group as ours hybridizing chips made by themselves or even utilizing
> chips spotted for them by a large microarray facility.

I'll have a dig around when I'm on the work computer with journal access 
rather than at home.  I've mentioned my four already (and will give full 
PMIDs if you want) - there are many more from our department alone, let 
alone other departments and other universities.  Though I don't know if we 
come under your (2) - how do you define a "large microarray facility" and/or 
a "large biomedical group"?

>
> I discovered these things :
> 1) The beginnig of the microarray experiment is high throughput . But
> the textminig like Onto express is still low throughput. There is no
> artificial intelligent software that extracts the cause of a desease
> from Pubmed with a list of regulated genes as input. The reason is ,
> ofcourse , that the information is not yet there.

Absolutely.  Moving from an observation of altered transcript levels to a 
biological explanation of the system you're investigating has a very high 
swearword:publication ratio.  In general you shouldn't expect to publish 
array data on its own, you'll need substantial follow-up work.  RT-PCR 
validation of the array hits, in situ studies to show expression 
distribution, detecting regulated pathways involving multiple genes - all 
takes a lot of work.  By the time you publish, the array results are a small 
proportion of the paper.  This may be why you're having trouble finding them 
on PubMed: most papers don't simply publish array data on its own, and those 
which do publish array data on its own tend as you saw to be mainly 
technical ones covering the array process itself.

>
> 2)This departement does skin research for the last 30 years but we
> never heard of the highest regulated genes we found , not much was
> known about them in Pubmed.

Did the results check out when you did RT-PCR followup, in situ hybs etc.? 
If so, then congratulations on the really novel findings!  This is another 
issue with array approaches - since they may lead you to areas well away 
from the beaten track, it's often much harder to fit the data into a 
coherent narrative to explain what's going on biologically.  This is not a 
problem with the array approach, it's a feature, not a bug.  I'd much rather 
take steps towards knowing what's *really* going on, rather than being 
constrained by whatever preconceptions have been built into the field 
already.

>
> We greatly underestimated the technical difficulties , the time and
> money needed for a microarray experiment . If I had to do this again
> I would give my purified RNA to a microarray facility that uses Affy
> chips and waited for the annotated list of regulated genes.Then I
> would start with that list . This would save me 80 % of the time and
> the cost of the project .

A useful approach for those for whom Affy is available, yes.  If it's not 
available for your organism, or you can't afford the comparatively large 
cost of Affy chips, then there *is* a role for getting your own bespoke 
chips printed.  However, get them printed by someone who really knows how.

>
> My sincere advice :
> If you are a small group of biologists and you want do to microarrays
> then look for a microarray facility at your university . Start
> talking to the bioinformatics and statistics guys even before you
> fill the first eppendorf . Work as closely together as you can .If
> they offer the service than give them your purified RNA and just wait
> for the annotated list of regulated genes. Than start from there. If
> your university does not have a large microarray facility look for a
> commercial solution.

I think I could agree with most of that.  I'd rather do the arrays myself 
rather than getting someone else to do them, but that I suppose is because 
I've been doing them for a while now and am familiar with the process.

Peter