From centolam at omrf.ouhsc.edu Thu Jul 4 18:05:41 2002 From: centolam at omrf.ouhsc.edu (Michael Centola) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] Chips are available Message-ID: <3D24D4C5.5050605@omrf.ouhsc.edu> Hi All, The OMRF microarray facility has mouse and human microarrays available and custom biostatistical techniques for analysis. We are producing about 100 chips every 2-3 weeks and we are looking for collaborators. If you have a mouse or human microarray project in mind please contact us at: Centolam@omrf.ouhsc.edu Here's a description of the human chips that I just put into a grant: Our facility has developed next-generation genome-scale oligo-based microarray production and hybridization technology. These arrays are produced using a commercially available library of 70 base pair long DNA oligos (70-mers, Qiagen/Operon Technologies). The oligos were derived from the functionally defined genes present in the UniGene and RefSeq databases (http://www.ncbi.nlm.nih.gov). Each oligo corresponds to a unique region within or proximal to the 3'end of a given gene. Length and sequence specificity have therefore been optimized, reducing or eliminating the cross-hybridization problems encountered with cDNA-based arrays. Our human arrays have 21,329 human genes represented. The library utilized for array production is therefore among the most comprehensive commercially available set of array-ready functionally defined human genes with all known human genes described prior to Dec. 2001 and over 10,000 undefined human cDNAs represented. These chips allow us to comprehensively identify key differences in experimental and control samples. A complete listing of the genes can be seen at the following web address:http://www.operon.com/arrays/humangenome.prn. Our two research and development scientists have been collaborating with Qiagen/Operon to enhance the dynamic range and signal-to-noise ratios of these oligo-based arrays. Our oligo microarray production and processing method is now distributed by Qiagen/Operon to microarray labs setting up to use oligo array technology. In addition, we are preparing a manuscript describing the development of our production and processing methods. The mouse chips are similarly comprehensive. They have 16,463 gene represented as 70mer oligo probes. A list of genes can be found at: http://www.operon.com/arrays/mouse_V2_384.prn The oligos are designed from the UniGene Database Build Mm 102 (February 2002) and the Mouse Reference Sequence (RefSeq) Database, both developed and maintained at the National Center of Biotechnology Information (NCBI) www.ncbi.nlm.nih.gov . We also have a team of bioinformatics scientists that can analyze the data from microarray experiments. Analysis is based on the research of Dr. Igor Dozmorov and includes differential gene expression and molecular pathway identification. Also in the group are Nicholas Knowlton, Parima Pathipvanich, and Alan Shields. These scientists incorporate our analytical methods into user-friendly software and are developing an integrated database system for datamining among clinical, genetic, and gene expression data sets. Mike Centola OMRF microarray facility director centolam@omrf.ouhsc.edu -------------- next part -------------- An HTML attachment was scrubbed... URL: http://lists.onenet.net/pipermail/okmicroarray/attachments/20020704/0e2944dd/attachment.htm From targetemailextractor at btamail.net.cn Sat Jul 20 00:48:22 2002 From: targetemailextractor at btamail.net.cn (targetemailextractor@btamail.net.cn) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] ADV: Direct email blaster, email addresses extractor, maillist verify, maillist manager........... Message-ID: <200207200616.g6K6GhT14855@lists.onenet.net> An HTML attachment was scrubbed... URL: http://lists.onenet.net/pipermail/okmicroarray/attachments/20020720/36e4ecd2/attachment.htm From centolam at omrf.ouhsc.edu Mon Aug 19 23:18:22 2002 From: centolam at omrf.ouhsc.edu (Michael Centola) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] wanted: gene expression profiling database Message-ID: <3D61C30E.1080108@omrf.ouhsc.edu> This email just arrived on the UCSF microarray listserve and I thought it might be useful to those of us building functional databases for gene expression profiling experiments (see below). By the way we are building a functional database for for mouse and human gene expression profiling experiments at the OMRF microarray research lab and we could use the guidance and collaboration of those interested. The project is the main responsibility of Alan Shields in my lab (along with Parima Pathipvanich, Igor Dozmorov, Bart Frank, Mollie Rangnow, and Nick Knowlton). We are building it on a Dell Server running MYSQL as the database platform and BASE ver 1.0 as the expression profile database. We will integrate gene ontogeny (gene function), chromosomal location, multiple names, cross reference between the genes of known function on our mouse and human chips (the RefSeq members), our analysis methods, as well as other forms of data (clinical or genetic) on the database. We will also be taking the time to set up a distributed computing network to handle some of the more complex analyses. If you have comments regarding this work or want to help or collaborate contact me (centolam@omrf.ouhsc.edu) or better yet contact someone in the lab who actually knows what they are doing in this regard, Alan Shields (shields@omrf.ouhsc.edu). Mike Centola -------- Original Message -------- Subject: Inhouse genomic databases and accession id maps Date: Mon, 19 Aug 2002 16:12:02 -0700 From: Kasian Franks Reply-To: "To share info regarding GeneChips tech. and Microarrays in general" To: GENE-ARRAYS@ITSSRV1.UCSF.EDU I have a list of databases I've built that are portable and available for inhouse installlation located here: http://64.163.147.146/~kasian/fp/databases.html If interested let me know and I'll send you one. __________________________________________________ Do You Yahoo!? HotJobs - Search Thousands of New Jobs http://www.hotjobs.com _____________________________________________ You may leave the list at any time by sending a message- "SIGNOFF GENE-ARRAYS" To: LISTSERV@ITSSRV1.UCSF.EDU NOTE: The message line should be in the body of the message and no where else, i.e. the subject line. -------------- next part -------------- An HTML attachment was scrubbed... URL: http://lists.onenet.net/pipermail/okmicroarray/attachments/20020819/5333cd74/attachment.htm From Alan-Shields at omrf.ouhsc.edu Tue Aug 20 15:35:39 2002 From: Alan-Shields at omrf.ouhsc.edu (Alan Shields) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] wanted: gene expression profiling database addendum Message-ID: <1029875739.2335.1.camel@fatman> Whoops. Wrong address for me in that e-mail. My REAL e-mail address is Alan-Shields@omrf.ouhsc.edu. Thanks, Alan From centolam at omrf.ouhsc.edu Thu Aug 29 01:32:03 2002 From: centolam at omrf.ouhsc.edu (Michael Centola) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] MIcroarray papers and grants Message-ID: <3D6DBFE3.4050707@omrf.ouhsc.edu> Hi all, I am updating a talk on the state of OK in the state. Can you update me on any new grants obtained and papers submitted or published that use microarrays this year (2002)? I have materials from 2001. thanks, Mike Centola centolam@omrf.ouhsc.edu From centolam at omrf.ouhsc.edu Sat Sep 7 09:01:19 2002 From: centolam at omrf.ouhsc.edu (Michael Centola) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] I thought it was Ozone Message-ID: <3D7A06AF.5020308@omrf.ouhsc.edu> Hi All, here's an interesting posting on Ozone and Cy5 and a Cy5 anti-fade reagent. Does anyone know if Oklahoma has high Ozone levels anytime of the year? Mike All, Per Ron Hart's request I would like to share some of the things we have learned about problems with fluorescent dyes on microarrays as well as provide additional information about our DyeSaver product. Over the last several years our technical support group has fielded quite a few calls regarding Cy5 failure on microarrays. This summer has been very active and we decided to take a look through our records as well as the archives of the List serv. One of the things we noticed is that the number of Tech calls and postings is highest during the time between early June and the end of August. It appears that Cy5 is more sensitive during the summer months. After a more detailed review we were able to subdivide the problems into 3 categories. 1: Little or no Cy5 signal because of either bad RNA or a poor cDNA synthesis. This is usually identified in a dye flip experiment and accounts for only a few of the problems reported. 2: Cy5 damage during the hybridization or washes. The source of the problem appears to be some substance that is either residual on the array or is part of the buffers. Sometime back we added our antifade reagent to our kits to prevent problems during the hybridization stage. However, this reagent does not eliminate problems during the washes. As posted, adding DTT to the wash buffer can help to minimize the problem but may not work for all cases. Typically we recommend that the water source be changed. Most of us assume that our water is not a problem in most assays and I think that most of the time the assumption is true. But it has been documented on the list serv, as well as has been confirmed by our Technical Support group, that changing water source has helped to eliminate some Cy5 degradation problems (due to the water used in buffers). We have specifically identified, as have others, that MilliQ water changes over time. Our best estimate is that once new cartridges are put onto a MilliQ (and maybe other DI units) there is a 3 month time frame where no problems will be observed. About 3 months after the cartridges are changed the Cy5 problems return. We do not have any idea what the agent causing the problem in the water is yet, but changing the water or adding DTT appears to help. We have qualified reagent grade DI from VWR. It comes in 20 liter boxes, is cheap, and we have not experienced any problems with Cy5 from something in the water for the past year when using this source. We did see problems with Cy5 when we used our MilliQ system even though the MilliQ unit indicated that everything was functionally appropriate. This is hard to believe and it has taken 3 years to convince myself that the water may contribute to Cy5 degradation. 3: Cy5 damage/fading as a result of oxidation or photobleaching in air, on the scanner, or in light. I have collected these causes together because we believe that each is due to a similar chemical process and because our DyeSaver reagent prevents the loss of Cy5 signal in all of these cases. Let me explain. When we first started working with Cy5 and Alexa 647 (both behave chemically similar with respect to fading on arrays), we observed that radicals will destroy these dyes. Throughout this summer we were continuously receiving ozone warnings in our area. Ozone forms radicals and we postulated that at high enough levels would destroy Cy5. So we ran a little experiment using an ozone generator. We hybridized several arrays with Cy3 and Cy5 labeled cDNA and scanned all of them to get a baseline signal. We then put half of them in a box with the ozone generator for 2-5 minutes and rescanned all of the slides after the process. The Cy5 signal on all of the arrays exposed to ozone was gone, but the Cy5 signal on the controls remained intact. After discussing the results with the group, we decided that we needed to develop a compound that we could use to protect the Cy5 from degradation in the ozone box. So we put together a variety of formulations of reagents that we thought might do the job and started testing the mixture just as a drug developer would screen new drug candidates, trial and error. Eventually we hit the right formulation that met the requirements and, as Ron Hart discussed, passes the ozone challenge. The requirements included: protects the Cy5 from ozone exposure, elicits no background, and works on a variety of surface chemistries. The end result was DyeSaver, which is a proprietary, patent pending reagent that can be applied to any array to preserve the dyes. It can be used with any labeling method (i.e. Genisphere, direct incorporation, amino allyl, etc.) The reagent is formulated as a liquid but, for convenience to the end user and to avoid waste, is applied as an aerosol spray to the slide. It literally takes a second to apply, and we have observed a number of additional benefits during our testing. First, when using our ScanArray5000 Scanner, normally we have to limit the Cy5 laser to less than 80% power and usually we get one shot at scanning the array because the laser "burns" the dye. However, with DyeSaver we have been able to set the laser at 100% and can scan the array repeatedly with little loss of Cy5 signal. Some other sites have confirmed this observation on their Scanarray scanner. Second, it appears that applying DyeSaver protects the array from exposure to light, direct sunlight. We ran an experiment with several arrays similar to that described above when testing the ozone generator. We hybridized the arrays with Cy3 and Cy5, did a baseline scan, and we sprayed some of them with the DyeSaver. We then placed the arrays outside in our parking lot for over 3 hours in direct sun. We also included some arrays with "no sun" exposure in a shaded box outside as a control. The temperature at the time of the experiment was in the mid 80s. Ozone levels were low per the National Weather Service. We then scanned all of the arrays and observed that the arrays in the direct sunlight that were NOT sprayed had lost all of their Cy5 signal and greater than 95% of their Cy3 signal. The sprayed arrays in direct sunlight had lost about 20-25% of their Cy5 signal and less than 5% of their Cy3 signal. The shaded arrays without spray had lost about 30% of their Cy5 signal and the Cy3 signal was unchanged. The sprayed arrays maintained in the shade were essentially unchanged. Based on our results to date the DyeSaver will be a good way to stabilize the dyes and may also be a nice way to preserve an array or other fluorescent assays in archive to be (re)scanned at a later date. We have studyied arrays that were sprayed with DyeSaver about a month ago and they have maintained their signal for both Cy3 and Cy5. No longer do you have to "rush to scan" an array. The experiments I discussed in this posting as well as some additional data will be posted on our webpage and will be included in a poster at the Chips to Hits conference in October in Philadelphia. We expect to be selling DyeSaver in November, and are currently providing a beta version to customers with Cy5 problems. The exact price of DyeSaver has not yet been determined, but it will be less than 25 cents per array. Each can will have enough material for about 250-300 arrays depending on the user. Anyone interested can email me at bob_getts@datascope.com or should contact us via the internet at www.genisphere.com. Bob Getts Manager R&D Genisphere Inc. ----- Original Message ----- From: Ron Hart To: Sent: Friday, August 30, 2002 10:08 AM Subject: Re: labelling Problems? > Hate to beat a dead horse, but we did try the Alexa dye comparable to Cy5 > and saw similar fading problems. > > However, I recently had the opportunity to see a new product that seems to > completely solve the dye fade problem. Bob Getts of Genisphere visited my > lab last week and brought a "pre-release" can of stuff he calls "Dye Saver." > It's a spray that is supposed to interfere with oxidation by ozone or other > radicals. Smells like organic solvents. > > The stuff worked perfectly. We prepared two arrays using identical probes > side-by-side, and Bob sprayed one of them with Dye Saver. Within minutes, > we could see the Cy5 fading in the unsprayed slide, and no change in the > sprayed slide. After a hour the difference was VERY obvious (the unsprayed > chip was almost totally green). Finally, we put both chips into a box with > an ozone generator to blow away the rest of the Cy5. The sprayed chips > survived with bright reds intact. > > After all the troubles we've had, this finally looks like a real solution. > In fact, if I had seen the images in sales literature I would have found it > hard to believe. But the results were indisputible. > > Perhaps Bob will be able to contribute a "Commercial" response with more > information? In the meantime, I hope my sample can of Dye Saver doesn't run > out before it goes on the market. > > Ron Hart > Rutgers University > > P.S. I have no financial interest in Genisphere. Just a happy customer. > > ----- Original Message ----- > From: "Phillips Jonathan M" > To: > Sent: Wednesday, August 28, 2002 11:26 AM > Subject: Re: labelling Problems? > > > > Dear List, > > > > I am somewhat amazed by all of the e-mail traffic with respect to Cy5. > > Virtually everything that I have seen (and heard) is negative. We have > been > > using Alexa 546 and Alexa 647 for about a year and a half and during that > > time, have never experienced any of the problems that have been described > in > > subscriber e-mails. The obvious question is "Why don't you switch to > Alexa > > dyes"? > > > > Cheers! > > > > Jonathan > > > > Jonathan M. Phillips, Ph.D. > > > > Molecular Pathologist > > Life Sciences Operation > > IIT Research Institute > > 10 West 35th Street > > Chicago, Il 60616-3793 > > Tel: 312-567-4217 > > Fax: 312-567-4852 > > > > _____________________________________________ > > > > You may leave the list at any time by sending a message- > > > > "SIGNOFF GENE-ARRAYS" > > > > To: LISTSERV@ITSSRV1.UCSF.EDU > > > > NOTE: The message line should be in the body of the message and no where > else, i.e. the subject line. > > > > _____________________________________________ > > You may leave the list at any time by sending a message- > > "SIGNOFF GENE-ARRAYS" > > To: LISTSERV@ITSSRV1.UCSF.EDU > > NOTE: The message line should be in the body of the message and no where else, i.e. the subject line. > _____________________________________________ You may leave the list at any time by sending a message- "SIGNOFF GENE-ARRAYS" To: LISTSERV@ITSSRV1.UCSF.EDU NOTE: The message line should be in the body of the message and no where else, i.e. the subject line. _____________________________________________ You may leave the list at any time by sending a message- "SIGNOFF GENE-ARRAYS" To: LISTSERV@ITSSRV1.UCSF.EDU NOTE: The message line should be in the body of the message and no where else, i.e. the subject line. -------------- next part -------------- An HTML attachment was scrubbed... URL: http://lists.onenet.net/pipermail/okmicroarray/attachments/20020907/68efb9b8/attachment.htm From liulin at cvm.okstate.edu Sun Sep 8 15:27:18 2002 From: liulin at cvm.okstate.edu (Lin Liu/phsi/Cvm) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] seminar at OSU Message-ID: Dr. Mala R. Chinoy, Associate Professor of Surgery, Penn State University will present a seminar entitled "cDNA Microarray Analysis of Cardiopulmonary Development Defects". Time: 3:30 pm, Sept 19, 2002 Place: 259 McElroy Hall, College of Veterinary Medicine, Oklahoma State University Dr. Chinoy will be available to meet with faculty at 8:30 am to 3:00 pm, Sept 19 or 8:30 am to 11:30 am, Sept 20. Let me know if everyone is interested. _____________________________ Lin Liu, Ph.D. Associate Professor Department of Physiological Sciences Oklahoma State University Stillwater, OK 74078 Tel: 405-744-4526 Fax: 405-744-8263 E-mail: liulin@okstate.edu From cmc at chappelllawfirm.com Sun Sep 8 08:41:58 2002 From: cmc at chappelllawfirm.com (Cherie M. Chappell) Date: Tue Mar 23 20:25:28 2004 Subject: [Okmicroarray] RE: ozone - Okmicroarray digest, Vol 1 #14 - 1 msg In-Reply-To: <200209071701.g87H1Am01324@lists.onenet.net> Message-ID: <007c01c2573d$814ddb20$6901a8c0@Edmondhome> Oklahoma (and OKC in particular) frequently has high levels of ozone. There are monitoring stations scattered throughout the state and in various neighborhoods in OKC and Tulsa. Oklahoma DEQ (dept of environmental quality) keeps an eye on air quality and has daily on-line maps of ozone levels and concentrations. http://www.mesonet.org/ozone/ See also, http://www.deq.state.ok.us/AQDnew/ Cherie Chappell > -----Original Message----- > From: okmicroarray-admin@lists.onenet.net > [mailto:okmicroarray-admin@lists.onenet.net]On Behalf Of > okmicroarray-request@lists.onenet.net > Sent: Saturday, September 07, 2002 12:01 PM > To: okmicroarray@lists.onenet.net > Subject: Okmicroarray digest, Vol 1 #14 - 1 msg > > > Send Okmicroarray mailing list submissions to > okmicroarray@lists.onenet.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://lists.onenet.net/mailman/listinfo/okmicroarray > or, via email, send a message with subject or body 'help' to > okmicroarray-request@lists.onenet.net > > You can reach the person managing the list at > okmicroarray-admin@lists.onenet.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Okmicroarray digest..." > > > The Oklahoma Microarray Question and Answer Forum > > > Today's Topics: > > 1. I thought it was Ozone (Michael Centola) > > --__--__-- > > Message: 1 > Date: Sat, 07 Sep 2002 09:01:19 -0500 > From: Michael Centola > To: okmicroarray@lists.onenet.net > Subject: [Okmicroarray] I thought it was Ozone > > > --------------060702010103050802020601 > Content-Type: text/plain; charset=us-ascii; format=flowed > Content-Transfer-Encoding: 7bit > > Hi All, > > here's an interesting posting on Ozone and Cy5 and a Cy5 anti-fade > reagent. Does anyone know if Oklahoma has high Ozone levels > anytime of > the year? > > Mike > > > All, > > Per Ron Hart's request I would like to share some of the > things we have > learned about problems with fluorescent dyes on microarrays as well as > provide additional information about our DyeSaver product. > Over the last > several years our technical support group has fielded quite a > few calls > regarding Cy5 failure on microarrays. This summer has been > very active and > we decided to take a look through our records as well as the > archives of > the List serv. One of the things we noticed is that the > number of Tech > calls and postings is highest during the time between early > June and the > end of August. It appears that Cy5 is more sensitive during > the summer > months. After a more detailed review we were able to subdivide the > problems into 3 categories. > > 1: Little or no Cy5 signal because of either bad RNA or a poor cDNA > synthesis. This is usually identified in a dye flip experiment and > accounts for only a few of the problems reported. > > 2: Cy5 damage during the hybridization or washes. The source of the > problem appears to be some substance that is either residual > on the array > or is part of the buffers. Sometime back we added our > antifade reagent to > our kits to prevent problems during the hybridization stage. > However, this > reagent does not eliminate problems during the washes. As > posted, adding > DTT to the wash buffer can help to minimize the problem but > may not work > for all cases. Typically we recommend that the water source > be changed. > Most of us assume that our water is not a problem in most assays and I > think that most of the time the assumption is true. But it has been > documented on the list serv, as well as has been confirmed by > our Technical > Support group, that changing water source has helped to > eliminate some Cy5 > degradation problems (due to the water used in buffers). We have > specifically identified, as have others, that MilliQ water > changes over > time. Our best estimate is that once new cartridges are put > onto a MilliQ > (and maybe other DI units) there is a 3 month time frame > where no problems > will be observed. About 3 months after the cartridges are > changed the Cy5 > problems return. We do not have any idea what the agent causing the > problem in the water is yet, but changing the water or > adding DTT appears > to help. We have qualified reagent grade DI from VWR. It comes in 20 > liter boxes, is cheap, and we have not experienced any > problems with Cy5 > from something in the water for the past year when using this > source. We > did see problems with Cy5 when we used our MilliQ system even > though the > MilliQ unit indicated that everything was functionally > appropriate. This > is hard to believe and it has taken 3 years to convince > myself that the > water may contribute to Cy5 degradation. > > 3: Cy5 damage/fading as a result of oxidation or > photobleaching in air, > on the scanner, or in light. I have collected these causes together > because we believe that each is due to a similar chemical process and > because our DyeSaver reagent prevents the loss of Cy5 signal in all of > these cases. Let me explain. When we first started working > with Cy5 and > Alexa 647 (both behave chemically similar with respect to fading on > arrays), we observed that radicals will destroy these dyes. > Throughout > this summer we were continuously receiving ozone warnings in our area. > Ozone forms radicals and we postulated that at high enough > levels would > destroy Cy5. So we ran a little experiment using an ozone > generator. We > hybridized several arrays with Cy3 and Cy5 labeled cDNA and > scanned all of > them to get a baseline signal. We then put half of them in a > box with the > ozone generator for 2-5 minutes and rescanned all of the > slides after the > process. The Cy5 signal on all of the arrays exposed to > ozone was gone, > but the Cy5 signal on the controls remained intact. After > discussing the > results with the group, we decided that we needed to develop > a compound > that we could use to protect the Cy5 from degradation in the > ozone box. So > we put together a variety of formulations of reagents that we > thought might > do the job and started testing the mixture just as a drug > developer would > screen new drug candidates, trial and error. Eventually we > hit the right > formulation that met the requirements and, as Ron Hart > discussed, passes > the ozone challenge. The requirements included: protects > the Cy5 from > ozone exposure, elicits no background, and works on a variety > of surface > chemistries. The end result was DyeSaver, which is a > proprietary, patent > pending reagent that can be applied to any array to preserve > the dyes. It > can be used with any labeling method (i.e. Genisphere, direct > incorporation, amino allyl, etc.) The reagent is formulated > as a liquid > but, for convenience to the end user and to avoid waste, is > applied as an > aerosol spray to the slide. It literally takes a second to > apply, and we > have observed a number of additional benefits during our > testing. First, > when using our ScanArray5000 Scanner, normally we have to > limit the Cy5 > laser to less than 80% power and usually we get one shot at > scanning the > array because the laser "burns" the dye. However, with > DyeSaver we have > been able to set the laser at 100% and can scan the array > repeatedly with > little loss of Cy5 signal. Some other sites have confirmed this > observation on their Scanarray scanner. Second, it appears > that applying > DyeSaver protects the array from exposure to light, direct > sunlight. We > ran an experiment with several arrays similar to that > described above when > testing the ozone generator. We hybridized the arrays with > Cy3 and Cy5, > did a baseline scan, and we sprayed some of them with the > DyeSaver. We > then placed the arrays outside in our parking lot for over 3 hours in > direct sun. We also included some arrays with "no sun" exposure in a > shaded box outside as a control. The temperature at the time of the > experiment was in the mid 80s. Ozone levels were low per the National > Weather Service. We then scanned all of the arrays and > observed that the > arrays in the direct sunlight that were NOT sprayed had lost > all of their > Cy5 signal and greater than 95% of their Cy3 signal. The > sprayed arrays in > direct sunlight had lost about 20-25% of their Cy5 signal and > less than 5% > of their Cy3 signal. The shaded arrays without spray had > lost about 30% of > their Cy5 signal and the Cy3 signal was unchanged. The sprayed arrays > maintained in the shade were essentially unchanged. > > Based on our results to date the DyeSaver will be a good way > to stabilize > the dyes and may also be a nice way to preserve an array or other > fluorescent assays in archive to be (re)scanned at a later > date. We have > studyied arrays that were sprayed with DyeSaver about a month > ago and they > have maintained their signal for both Cy3 and Cy5. No longer > do you have > to "rush to scan" an array. > > The experiments I discussed in this posting as well as some > additional data > will be posted on our webpage and will be included in a > poster at the Chips > to Hits conference in October in Philadelphia. We expect to > be selling > DyeSaver in November, and are currently providing a beta version to > customers with Cy5 problems. The exact price of DyeSaver has > not yet been > determined, but it will be less than 25 cents per array. > Each can will > have enough material for about 250-300 arrays depending on the user. > Anyone interested can email me at bob_getts@datascope.com or > should contact > us via the internet at www.genisphere.com. > > > Bob Getts > Manager R&D > Genisphere Inc. > > > > > > > ----- Original Message ----- > From: Ron Hart > To: > Sent: Friday, August 30, 2002 10:08 AM > Subject: Re: labelling Problems? > > > > Hate to beat a dead horse, but we did try the Alexa dye > comparable to Cy5 > > and saw similar fading problems. > > > > However, I recently had the opportunity to see a new > product that seems to > > completely solve the dye fade problem. Bob Getts of > Genisphere visited my > > lab last week and brought a "pre-release" can of stuff he calls "Dye > Saver." > > It's a spray that is supposed to interfere with oxidation > by ozone or > other > > radicals. Smells like organic solvents. > > > > The stuff worked perfectly. We prepared two arrays using > identical probes > > side-by-side, and Bob sprayed one of them with Dye Saver. > Within minutes, > > we could see the Cy5 fading in the unsprayed slide, and no > change in the > > sprayed slide. After a hour the difference was VERY obvious (the > unsprayed > > chip was almost totally green). Finally, we put both chips > into a box > with > > an ozone generator to blow away the rest of the Cy5. The > sprayed chips > > survived with bright reds intact. > > > > After all the troubles we've had, this finally looks like a > real solution. > > In fact, if I had seen the images in sales literature I > would have found > it > > hard to believe. But the results were indisputible. > > > > Perhaps Bob will be able to contribute a "Commercial" > response with more > > information? In the meantime, I hope my sample can of Dye > Saver doesn't > run > > out before it goes on the market. > > > > Ron Hart > > Rutgers University > > > > P.S. I have no financial interest in Genisphere. Just a > happy customer. > > > > ----- Original Message ----- > > From: "Phillips Jonathan M" > > To: > > Sent: Wednesday, August 28, 2002 11:26 AM > > Subject: Re: labelling Problems? > > > > > > > Dear List, > > > > > > I am somewhat amazed by all of the e-mail traffic with > respect to Cy5. > > > Virtually everything that I have seen (and heard) is > negative. We have > > been > > > using Alexa 546 and Alexa 647 for about a year and a half > and during > that > > > time, have never experienced any of the problems that have been > described > > in > > > subscriber e-mails. The obvious question is "Why don't > you switch to > > Alexa > > > dyes"? > > > > > > Cheers! > > > > > > Jonathan > > > > > > Jonathan M. Phillips, Ph.D. > > > > > > Molecular Pathologist > > > Life Sciences Operation > > > IIT Research Institute > > > 10 West 35th Street > > > Chicago, Il 60616-3793 > > > Tel: 312-567-4217 > > > Fax: 312-567-4852 > > > > > > _____________________________________________ > > > > > > You may leave the list at any time by sending a message- > > > > > > "SIGNOFF GENE-ARRAYS" > > > > > > To: LISTSERV@ITSSRV1.UCSF.EDU > > > > > > NOTE: The message line should be in the body of the > message and no where > > else, i.e. the subject line. > > > > > > > _____________________________________________ > > > > You may leave the list at any time by sending a message- > > > > "SIGNOFF GENE-ARRAYS" > > > > To: LISTSERV@ITSSRV1.UCSF.EDU > > > > NOTE: The message line should be in the body of the message > and no where > else, i.e. the subject line. > > > > _____________________________________________ > > You may leave the list at any time by sending a message- > > "SIGNOFF GENE-ARRAYS" > > To: LISTSERV@ITSSRV1.UCSF.EDU > > NOTE: The message line should be in the body of the message > and no where else, i.e. the subject line. > > _____________________________________________ > > You may leave the list at any time by sending a message- > > "SIGNOFF GENE-ARRAYS" > > To: LISTSERV@ITSSRV1.UCSF.EDU > > NOTE: The message line should be in the body of the message > and no where else, i.e. the subject line. > > > > --------------060702010103050802020601 > Content-Type: text/html; charset=us-ascii > Content-Transfer-Encoding: 7bit > > > > > content="text/html;charset=iso-8859-1"> > > > > Hi All,
>
> here's an interesting posting on Ozone and Cy5 and a Cy5 > anti-fade reagent. >  Does anyone know if Oklahoma has high Ozone levels > anytime of the year?
>
> Mike
>
> 
>
> All,
>
> Per Ron Hart's request I would like to share some of the
> things we have
> learned about problems with fluorescent dyes on microarrays as well as
> provide additional information about our DyeSaver product.
> Over the last
> several years our technical support group has fielded quite a
> few calls
> regarding Cy5 failure on microarrays.  This summer has been
> very active and
> we decided to take a look through our records as well as the
> archives of
> the List serv.  One of the things we noticed is that the
> number of Tech
> calls and postings is highest during the time between early
> June and the
> end of August.  It appears that  Cy5 is more sensitive during
> the summer
> months.  After a more detailed review we were able to subdivide the
> problems into 3 categories.
>
> 1:  Little or no Cy5 signal because of either bad RNA or a poor cDNA
> synthesis.  This is usually identified in a dye flip experiment and
> accounts for only a few of the problems reported.
>
> 2:  Cy5 damage during the hybridization or washes.  The source of the
> problem appears to be some substance that is either residual
> on the array
> or is part of the buffers.  Sometime back we added our
> antifade reagent to
> our kits to prevent problems during the hybridization stage.
> However, this
> reagent does not eliminate problems during the washes.  As
> posted, adding
> DTT to the wash buffer can help to minimize the problem but
> may not work
> for all cases.  Typically we recommend that the water source
> be changed.
> Most of us assume that our water is not a problem in most assays and I
> think that most of the time the assumption is true.  But it has been
> documented on the list serv, as well as has been confirmed by
> our Technical
> Support group, that changing water source has helped to
> eliminate some Cy5
> degradation problems (due to the water used in buffers).  We have
> specifically identified, as have others, that MilliQ water
> changes over
> time.  Our best estimate is that once new cartridges are put
> onto a MilliQ
> (and maybe other DI units) there is a 3 month time frame
> where no problems
> will be observed.  About 3 months after the cartridges are
> changed the Cy5
> problems return.  We do not have any idea what the agent causing the
> problem in the water is yet,  but changing the water or
> adding DTT appears
> to help.  We have qualified reagent grade DI from VWR.  It comes in 20
> liter boxes, is cheap, and we have not experienced any
> problems with Cy5
> from something in the water for the past year when using this
> source.  We
> did see problems with Cy5 when we used our MilliQ system even
> though the
> MilliQ unit indicated that everything was functionally
> appropriate.  This
> is hard to believe and it has taken 3 years to convince
> myself that the
> water may contribute to Cy5 degradation.
>
> 3:  Cy5 damage/fading  as a result of oxidation or
> photobleaching in air,
> on the scanner, or in light.  I have collected these causes together
> because we believe that each is due to a similar chemical process and
> because our DyeSaver reagent prevents the loss of Cy5 signal in all of
> these cases.  Let me explain.  When we first started working
> with Cy5 and
> Alexa 647 (both behave chemically similar with respect to fading on
> arrays), we observed that radicals will destroy these dyes.
> Throughout
> this summer we were continuously receiving ozone warnings in our area.
> Ozone forms radicals and we postulated that at  high enough
> levels would
> destroy Cy5.  So we ran a little experiment using an ozone
> generator.  We
> hybridized several arrays with Cy3 and Cy5 labeled cDNA and
> scanned all of
> them to get a baseline signal.  We then put half of them in a
> box with the
> ozone generator for 2-5 minutes and rescanned all of the
> slides after the
> process.  The Cy5 signal on all of the arrays exposed to
> ozone was gone,
> but the Cy5 signal on the controls remained intact.  After
> discussing the
> results with the group, we decided that we needed to develop
> a compound
> that we could use to protect the Cy5 from degradation in the
> ozone box.  So
> we put together a variety of formulations of reagents that we
> thought might
> do the job and started testing the mixture just as a drug
> developer  would
> screen new drug candidates, trial and error.  Eventually we
> hit the right
> formulation that met the requirements and, as Ron Hart
> discussed, passes
> the ozone challenge.  The requirements included:  protects
> the Cy5 from
> ozone exposure, elicits no background, and works on a variety
> of surface
> chemistries.  The end result was DyeSaver, which is a
> proprietary, patent
> pending reagent that can be applied to any array to preserve
> the dyes.  It
> can be used with any labeling method (i.e. Genisphere, direct
> incorporation, amino allyl, etc.)  The reagent is formulated
> as a liquid
> but, for convenience to the end user and to avoid waste, is
> applied as an
> aerosol spray to the slide.   It literally takes a second to
> apply, and we
> have observed a number of additional benefits during our
> testing.   First,
> when using our ScanArray5000 Scanner, normally we have to
> limit the Cy5
> laser to less than 80% power and usually we get one shot at
> scanning the
> array because the laser "burns" the dye.  However, with
> DyeSaver we have
> been able to set the laser at 100% and can scan the array
> repeatedly with
> little loss of Cy5 signal.  Some other sites have confirmed this
> observation on their Scanarray scanner.  Second,  it appears
> that  applying
> DyeSaver protects the array from exposure to light, direct
> sunlight.  We
> ran an experiment with several arrays similar to that
> described above when
> testing the ozone generator.  We hybridized the arrays with
> Cy3 and Cy5,
> did a baseline scan,  and we sprayed some of them with the
> DyeSaver.   We
> then placed the arrays outside in our parking lot for over 3 hours in
> direct sun.  We also included some arrays with "no sun" exposure in a
> shaded box outside as a control.  The temperature at the time of the
> experiment was in the mid 80s.  Ozone levels were low per the National
> Weather Service.  We then scanned all of the arrays and
> observed that the
> arrays in the direct sunlight that were NOT sprayed had lost
> all of their
> Cy5 signal and greater than 95% of their Cy3 signal.  The
> sprayed arrays in
> direct sunlight had lost about 20-25% of their Cy5 signal and
> less than 5%
> of their Cy3 signal.  The shaded arrays without spray had
> lost about 30% of
> their Cy5 signal and the Cy3 signal was unchanged.  The sprayed arrays
> maintained in the shade were essentially unchanged.
>
> Based on our results to date the DyeSaver will be a good way
> to stabilize
> the dyes and may also be a nice way to preserve an array or other
> fluorescent assays in archive to be (re)scanned at a later
> date.  We have
> studyied arrays that were sprayed with DyeSaver about a month
> ago and they
> have maintained their signal for both Cy3 and Cy5.  No longer
> do you have
> to "rush to scan" an array.
>
> The experiments I discussed in this posting as well as some
> additional data
> will be posted on our webpage and will be included in a
> poster at the Chips
> to Hits conference in October in Philadelphia.  We expect to
> be selling
> DyeSaver in November,  and are currently providing a beta version to
> customers with Cy5 problems.  The exact price of DyeSaver has
> not yet been
> determined, but it will be less than 25 cents per array.
> Each can will
> have enough material for about 250-300 arrays depending on the user.
> Anyone interested can email me at  class="moz-txt-link-abbreviated"
> href="mailto:bob_getts@datascope.com">bob_getts@datascope.com<
> /a> or should contact
> us via the internet at  href="http://www.genisphere.com">www.genisphere.com.
>
>
> Bob Getts
> Manager R&D
> Genisphere Inc.
>
>
>
>
>
>
> ----- Original Message -----
> From: Ron Hart 
> To: 
> Sent: Friday, August 30, 2002 10:08 AM
> Subject: Re: labelling Problems?
>
>
> > Hate to beat a dead horse, but we did try the Alexa dye
> comparable to Cy5
> > and saw similar fading problems.
> >
> > However, I recently had the opportunity to see a new
> product that seems to
> > completely solve the dye fade problem.  Bob Getts of
> Genisphere visited my
> > lab last week and brought a "pre-release" can of stuff
> he calls "Dye
> Saver."
> > It's a spray that is supposed to interfere with
> oxidation by ozone or
> other
> > radicals.  Smells like organic solvents.
> >
> > The stuff worked perfectly.  We prepared two arrays
> using identical probes
> > side-by-side, and Bob sprayed one of them with Dye
> Saver.  Within minutes,
> > we could see the Cy5 fading in the unsprayed slide, and
> no change in the
> > sprayed slide.  After a hour the difference was VERY obvious (the
> unsprayed
> > chip was almost totally green).  Finally, we put both
> chips into a box
> with
> > an ozone generator to blow away the rest of the Cy5.
> The sprayed chips
> > survived with bright reds intact.
> >
> > After all the troubles we've had, this finally looks
> like a real solution.
> > In fact, if I had seen the images in sales literature I
> would have found
> it
> > hard to believe.  But the results were indisputible.
> >
> > Perhaps Bob will be able to contribute a "Commercial"
> response with more
> > information?  In the meantime, I hope my sample can of
> Dye Saver doesn't
> run
> > out before it goes on the market.
> >
> > Ron Hart
> > Rutgers University
> >
> > P.S. I have no financial interest in Genisphere.  Just a
> happy customer.
> >
> > ----- Original Message -----
> > From: "Phillips Jonathan M" 
> > To: 
> > Sent: Wednesday, August 28, 2002 11:26 AM
> > Subject: Re: labelling Problems?
> >
> >
> > > Dear List,
> > >
> > > I am somewhat amazed by all of the e-mail traffic
> with respect to Cy5.
> > > Virtually everything that I have seen (and heard)
> is negative.  We have
> > been
> > > using Alexa 546 and Alexa 647 for about a year and
> a half and during
> that
> > > time, have never experienced any of the problems
> that have been
> described
> > in
> > > subscriber e-mails.  The obvious question is "Why
> don't you switch to
> > Alexa
> > > dyes"?
> > >
> > > Cheers!
> > >
> > > Jonathan
> > >
> > > Jonathan M. Phillips, Ph.D.
> > >
> > > Molecular Pathologist
> > > Life Sciences Operation
> > > IIT Research Institute
> > > 10 West 35th Street
> > > Chicago, Il 60616-3793
> > > Tel: 312-567-4217
> > > Fax: 312-567-4852
> > >
> > > _____________________________________________
> > >
> > > You may leave the list at any time by sending a message-
> > >
> > >  "SIGNOFF GENE-ARRAYS"
> > >
> > > To:    href="mailto:LISTSERV@ITSSRV1.UCSF.EDU">LISTSERV@ITSSRV1.UCSF.EDU
> > >
> > > NOTE: The message line should be in the body of the
> message and no where
> > else, i.e. the subject line.
> > >
> >
> > _____________________________________________
> >
> > You may leave the list at any time by sending a message-
> >
> >  "SIGNOFF GENE-ARRAYS"
> >
> > To:    href="mailto:LISTSERV@ITSSRV1.UCSF.EDU">LISTSERV@ITSSRV1.UCSF.EDU
> >
> > NOTE: The message line should be in the body of the
> message and no where
> else, i.e. the subject line.
> >
>
> _____________________________________________
>
> You may leave the list at any time by sending a message-
>
>  "SIGNOFF GENE-ARRAYS"
>
> To:    href="mailto:LISTSERV@ITSSRV1.UCSF.EDU">LISTSERV@ITSSRV1.UCSF.EDU
>
> NOTE: The message line should be in the body of the message
> and no where else, i.e. the subject line.
>
> _____________________________________________
>
> You may leave the list at any time by sending a message-
>
>  "SIGNOFF GENE-ARRAYS"
>
> To:    href="mailto:LISTSERV@ITSSRV1.UCSF.EDU">LISTSERV@ITSSRV1.UCSF.EDU
>
> NOTE: The message line should be in the body of the message
> and no where else, i.e. the subject line.
>
>
> > > > --------------060702010103050802020601-- > > > > --__--__-- > > _______________________________________________ > Okmicroarray mailing list > Okmicroarray@lists.onenet.net > http://lists.onenet.net/mailman/listinfo/okmicroarray > > > End of Okmicroarray Digest