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Microarray Solutions
            The Nexterion® Newsletter                     Edition # 2 / September 2006




        ARRAY BASED COMPARATIVE GENOMIC
    HYBRIDIZATION – CURRENTLY THE HOTTEST TOPIC
                 IN MICROARRAYING
          “Microarray Solutions” reports on the increasing popularity of array-CGH in the microarray
            community, and highlights how some of the most important facilities in North America
                               are using Nexterion® slides for this application.
                                           …read more on pages 4 and 5


                                    Also featured this month...

                           page 3      INTERVIEW WITH JAMI LOMAX
                                       New Sales Manager for East Coast North America & Technical
                                       Support Specialist for North America

                           page 6      NEW NEXTERION® LOW EVAPORATION BUFFER
                                       How the Nexterion® LEB Kit can solve the problem of source plate evapora-
                                       tion during extended print runs

                           page 7      TECHNICAL SUPPORT IN FOCUS
                                       How SCHOTT technical support assisted The Institute for Genomic Research
                                       in the use of the LEB Kit with Nexterion® Slide E

                           page 8      LATEST NEWS FROM SCHOTT
                                       Including ISO 9001 certification for the SCHOTT Microarray Solutions unit,
                                       new surface chemistries for the HiSens range of Slides, and the Nexterion®
                                       Web shop




Microarray Solutions
SCHOTT North America Inc.
5530 Shepherdsville Road
Louisville, KY 40228
USA
Phone: +1- 502-657-4417
Fax:      +1- 502-966-4976
E-Mail: coatedsubstrate@us.schott.com
www.us.schott.com/nexterion
Microarray Solutions                                                                                                page 2



                                       General Manager Talk


                                   Dear Reader,

                                   Welcome to the second edition of “Microarray Solutions”, the newsletter from SCHOTT.
                                   The response from readers around the world to the first edition of the newsletter was
                                   extremely positive, and I would like to thank all readers who provided us with feedback.

                                   We are pleased to welcome a new member to the SCHOTT Microarray Solutions team
                                   in the U.S., Jami Lomax (see interview on Page 3). Jami joined our company in June
                                   and will be the Sales Manager covering the Eastern region of North America. She will
                                   also assume the role of Technical Support Specialist for North America. Jami will no
                                   doubt be in contact with our North American customers very soon, and she will be
                                   supported in her Technical Support role by Dr. Rajendra Redkar from our R&D site in
                                   Duryea, PA. Both Jami and Raj will continue to provide the first class technical support
                                   that SCHOTT is known for in the microarray community. They will also both feature
                                   prominently alongside our R&D Director, Dr. Ruediger Dietrich, in the popular Techni-
                                   cal Support in Focus section of the newsletter (see Page 7).

                                   In Asia, SCHOTT has established an extensive distribution network to offer Nexterion®
                                   microarray products into this rapidly growing market. SCHOTT already had a strong
                                   relationship with several key microarray facilities in Asia, but in recent months we have
                                   received an increasing number of enquiries from the Far East. To meet these requests,
                                   SCHOTT will be making full use of its international network of sales offices, as well as
                                   working in partnership with experienced third party companies who distribute life sci-
                                   ence products in the Far East. Further details on the new sales infrastructure for
                                   SCHOTT in Asia will be announced very soon.

                                   This edition of the “Microarray Solutions” features an article on Array CGH, an increas-
       Dr. Lutz Wehmeier           ingly popular application. The article gives an overview of this exciting application from
       General Manager             our perspective, and that of some of our customers. It also demonstrates how a variety
Nexterion – Microarray Solutions   of surface chemistries offered by SCHOTT Microarray Solutions have been success-
                                   fully utilized for Array CGH. We also include a very interesting article on the new Nex-
                                   terion® Low Evaporation Buffer (LEB) kit that was recently launched by SCHOTT. The
                                   feedback we have had so far on this product from customers has been excellent, and
                                   our article on TIGR is a prime example of how the LEB Kit can be used to resolve a
                                   very common problem encountered by microarray users.

                                   I hope you enjoy this edition of Microarray Solutions, and thank you for choosing
                                   SCHOTT as your preferred microarray substrate supplier.




                                   Regards,
                                   Lutz Wehmeier
Microarray Solutions                                                                                                  page 3



    SCHOTT Profile – Jami Lomax East Coast Sales Manager and Technical
                           Support North America
                                       MS: How long have you worked for SCHOTT? And where did you work prior to
                                       SCHOTT?
                                       JL: I joined SCHOTT as Sales and Support Manager in early June this year. Prior to
                                       SCHOTT I worked with Spectrumedix LLC as an account manager and applications
                                       support consultant. I was previously a research technician for SCHOTT Nexterion R&D
                                       in Duryea, PA where I was involved in the development of the most of the current
                                       slides in our product portfolio.
 “Microarray Solutions”                MS: What key skills are required to do your job?
 catches up with the most              JL: I think one of the key skills necessary to be a sales manager and technical support
 recent addition to the                consultant is organization, patience, and time management! Most importantly, how-
 SCHOTT Microarray Solu-               ever, is the ability to quickly identify the needs of customers and be able to communi-
 tions team.                           cate with them clearly and concisely.
                                       MS: What do you do in technical support?
                                       JL: As technical support manager for North America, I help to troubleshoot any prob-
                                       lems customers have working with our products and also offer advice and protocol
                                       recommendations to customers prior to their slide evaluations. This can be done via e-
                                       mail or over the phone, but I am also able to go on-site to help work through protocols
                                       and provide tips on how to get the most from Nexterion® products. In my previous role
                                       working in R&D at SCHOTT, I played a key part in the development of several of the
                                       Nexterion® products, including protocol optimization, so I feel very comfortable offering
                                       advice to customers based on first-hand experience with the products.
                                       MS: Can you describe your typical work day?
                                       JL: The nice thing about this job is that every day offers new challenges and no two
                                       days are ever the same! On the sales side, I spend a lot of time travelling to visit cus-
                                       tomers throughout my territory, talking to new potential customers and servicing exist-
                                       ing customers. For technical support, a question from customers can obviously arrive
                                       at any time and usually requires an immediate response, usually involving a telephone
                                       conversation with the customer.
                                       MS: How do you respond to customer queries?
                                       JL: My response to customer queries usually depends on the type of customer and the
Technical Support contacts:            nature of the problem. The more basic questions can easily be addressed by either e-
                                       mail or telephone. Other queries require a more detailed response. For example, we
North America                          usually offer customers the opportunity to discuss the SCHOTT products and protocols
Contact: Jami Lomax                    in detail prior to their slide evaluation via a conference call. Or for customers who are
E-mail: jami.lomax@us.schott.com       experiencing significant problems getting a product to work for them, I may even spend
                                       some time in the lab with them to offer the best advice possible.
Europe, Asia, Rest of the World        MS: What are the most important aspects of technical support?
Contact: Dr. Ruediger Dietrich         JL: In my opinion, and fortunately that of SCHOTT, technical support is one of the
E-mail: ruediger.dietrich@schott.com   most valuable things a company can offer, particularly with regards to microarrays
                                       where there are so many variables that can ultimately have an effect on results. We
                                       pride ourselves on responding to customer problems as quickly as possible to minimize
                                       down time. SCHOTT has developed an excellent reputation for providing first-class
                                       customer support, and I will strive to maintain this.
                                       MS: What part of the job do you find most satisfying?
                                       JL: I think the most satisfying aspect is undoubtedly resolving a particularly problem-
                                       atic customer query and feeling the sense of relief and gratitude from the customer.
                                       Many people I deal with are under extreme pressure to deliver results and being able
                                       to support them and contribute in some way to their success is a nice aspect of the job,
                                       particularly when you see how passionate and dedicated they are to their research.
                                       MS: What's special about Nexterion®'s technical support?
                                       JL: When interviewed for the position at SCHOTT, one of the major things that struck
                                       me was SCHOTT’s commitment to technical support. Dr. Lutz Wehmeier, the General
                                       Manager, was adamant that one of SCHOTT’s key advantages over competitors was
                                       our comprehensive technical support services, and he felt that this, coupled with the
                                       product quality, was a significant reason for SCHOTT making such a big impact in the
                                       microarray substrate market in such a short time. The knowledge base and experience
                                       within SCHOTT is a big bonus. We have staff who have over eight years experience of
                                       working with the Nexterion® slide coatings and this allows us to produce very high
                                       quality slides as well as quickly troubleshoot customer problems. Our technical support
                                       team is also structured in such a way that we can offer an immediate response to cus-
                                       tomers wherever they are in the world.
Microarray Solutions                                                                                                    page 4



   Customer Application Focus – Comparative Genomic Hybridization using
                             Nexterion® Coated Slides
                     SCHOTT Insert data / images
                         •




                                        Array-CGH Background
                                        Technological advances in the field of genomics have resulted in the sequencing of
                                        genomes from several organisms, including the human genome. The availability of
                                        genomic data has made it possible to screen genetic abnormalities on the genome-
                                        wide scale. Comparative genomic hybridization (CGH) is a conventional molecular
                                        cytogenetic method capable of detecting deletions, additions and amplification of DNA
                                        copy numbers1. However, one of the limitations of this method is lower sensitivity or
                                        spatial resolution in detecting single copy deletion. Array-CGH, which is based on
                                        microarray technology, dramatically increases the resolution and dynamic range for
                                        detection of deletions and amplification. For instance, copy number changes at the
                                        level of 5-10 kilobases of DNA sequences can be detected using array-CGH as against
                                        10 megabases using conventional CGH. In addition, array-CGH allows direct mapping
Fig. 1 Results from Combimatrix         of genetic aberrations on the chromosomes, does not require cell culture or metaphase
Molecular Diagnostics of an Array-      chromosome, and improves throughput of testing. As the array format lends itself to
CGH experiment with Nexterion®          automation, array-based CGH technology should soon be practical for diagnosis of
HiSens A slides from SCHOTT.            cancer or genetic diseases in the clinical setting.
CMDX intends to offer these arrays
for clinical diagnostic applications.
                                        Array-CGH consists of an orderly arrangement of human DNA probes derived from
                                        coding and non-coding regions on coated glass substrate2,3,4. The DNA can be pre-
                                        pared from BACs (Bacterial artificial chromosomes) amplified by PCR from cDNA li-
                                        braries or specific genomic sequences5. The probes are printed robotically on an ami-
                                        nosilane or other coated microarray slide2. Alternatively, Approaches such as genomic
                                        representation arrays6, oligonucleotide arrays7 and specific target arrays8 have been
                                        also successfully used. For typical array CGH analysis DNA from a test and normal
                                        reference sample are labelled with different fluorophores and co-hybridized to the ar-
                                        ray9. After hybridization the ratio of fluorescence intensity of test to reference DNA
                                        sample (log2 ratios) is calculated to measure the copy number changes. Samples that
Fig. 2 High density human BAC 20K       do not show any genetic aberrations will indicate a constant ratio across the genome
array printed on Schott Nexterion® A+   area. Therefore, array-CGH offers a flexible platform for studying genetic abnormalities
slides                                  at genome wide level, which can be performed in a high throughput, and cost-effective
                                        way.


                                        Array-CGH in Clinical Applications
                                        Array-CGH has been used as a research tool that can address many human health
                                        problems that involves genetic abnormalities including carcinomas and specific condi-
                                        tions such as Down Syndrome. Recently, several commercial companies are convert-
                                        ing this research tool into a diagnostic product by offering array-CGH products and
                                        services for clinical laboratories. These companies are currently seeking clarification of
                                        regulatory requirements for array-CGH and are in process of getting FDA approval for
                                        clinical applications.


                                        In order to meet FDA regulations, data generated from array-CGH assays needs to
                                        meet certain criteria and this is the challenge, for example, facing many companies
Data was kindly provided by Dr. Norma   looking to offer arrays to the clinical diagnostics community. The data should show low
Nowak, Roswell Park Cancer Institute
                                        levels of variability among the replicates, samples with identical copy numbers, and
                                        non-specific background. Therefore the protocol for producing array-CGH assays
                                        needs to be carefully optimized. Another key consideration in this process is the quality
                                        of coated microarray slide, as this is critical for optimal immobilization of the probes
                                        and their accessibility to the target for hybridization, as well as for obtaining a low hy-
                                        bridization background. The coated glass substrate not only influences these factors,
                                        but is also important in determining whether users can consistently generate repro-
                                        ducible results day in and day out. SCHOTT is one of the few slide manufacturers able
                                        to offer the high level of lot to lot reproducibility required for diagnostic type applica-
                                        tions.
Microarray Solutions                                                                                                        page 5




                                              Nexterion® Slide A and Slide A+ for array-CGH Applications
                                              The most popular slide surface chemistry for array-CGH has traditionally been Ami-
Fig. 3: Representative human 19K              nosilane, with Nexterion® Slide A and Slide A+ having performed exceptionally well for
BAC aCGH profile of melanoma tumor            this type of application at numerous facilities throughout the world. The Microarray and
demonstrating detection of DNA seg-           Genomics Facility at Roswell Park Cancer Research Institute (RPCI) in Buffalo, NY is
ment copy number changes. Average             one of SCHOTT’s oldest North American customers, having successfully used both
log2 ratios (y-axis) were plotted for all     Slide A and Slide A+ for an array-CGH application with a slightly modified version of
clones based on chromosomal posi-             the recommended SCHOTT protocols. Currently they use Nexterion® Slide A+ slides to
tion (x-axis) with the blue bar demar-        print their high-density, and Nexterion® Slide A to print their lower density (<15k) BAC
cating the chromosomes (top plot) and         arrays. The arrayed products have excellent morphology and retention on both slide
centromeres (bottom plots). Horizontal        surfaces. Slide A+ allows RPCI to print over 70,000 features per slide thanks to the
red lines indicate the log2 ratio for         hydrophobic nature of the surface chemistry. SCHOTT’s state-of-the-art slide produc-
each segment as segmented by Circu-           tion facility and experienced production team in Germany, manufacture slides with a
                                              very high lot-to-lot consistency, helping to minimise variance between print runs, and
lar Binary Segmentation (CBS).
                                              giving the RPCI facility the confidence to use these slides for research projects lasting
                                              several years.

  Figure 1: High density human BAC array
  printed on Schott Nexterion type A+         The protocol followed by Roswell is relatively simple, with minimal post-print process-
  slides.                                     ing required for either of the Nexterion® Aminosilane slide surfaces after spotting BAC
  The figure represents an overlay pseudo-    printing solutions. In the RPCI facility, the arrayed slides are stored in a desiccated
  color image of a RPCI-119
           Chr 5       Chr BAC microar- 17
                                      Chr     environment after printing and UV cross linked immediately prior to hybridization. Suc-
  ray, co-hybridized with fluorescently       cinic anhydride blocking, boiling and/or SDS washes did not improve the performance
  labeled DNA derived from a melanoma
  sample (Cy5™) and a 5
                    Chr pooled human          of the slides, but in fact added background and other reagent related artefacts that can
  reference control (Cy3™). LM-PCR            be detected after scanning. The success achieved in the RPCI facility using SCHOTT
  representations of 19,000 BAC DNAs          Nexterion® slides has led to the decision to utilize SCHOTT Nexterion® slides exclu-
  were spotted in duplicate on Nexterion®     sively to manufacture BAC arrays for Empire Genomics, a spin-off company from RPCI
  Slide A+ using a BioRobotics MicroGrid II
  TAS arrayer and MicroSpot 10K split pins    recently launched by Dr. Norma Nowak. Dr. Nowak commented, “We are successfully
  (Genomic Solutions, Inc. Ann Arbor, MI)     using SCHOTT Nexterion® slides in our BAC aCGH services division of Empire Ge-
  at 150 µm spacing. Competitive hybridi-     nomics. This product enables us to analyze thousands of test DNAs and to reliably
  zation of the melanoma sample with the      compare their copy number status to control samples. When it comes to printing thou-
  reference control Chr 9 copy number
                    identifies
  amplifications (red spots) and deletions    sands of BAC elements at high density over thousands of slides, Nexterion® Slide A+
  (green spots).                              from SCHOTT is clearly the best slide out there.” (Fig. 3)


                                              Other SCHOTT slides successfully used for array-CGH
                                              In North America., SCHOTT has been receiving an increasing number of enquiries
                  Chr 17                      from the microarray community for Nexterion® coated substrates for array CGH. Com-
                                              bimatrix Molecular Diagnostics (Nexterion® Slide A) (Fig. 1), UCSF Comprehensive
                                              Cancer Center (Slide A, A+ and E) and BC Cancer Agency (Slide AL) have all been
                                              successful in using Nexterion® substrates for array CGH with BAC clones. Stanford
                                              University has produced arrays for CGH using Invitrogen’s HEEBO (Human Exonic
                                              Evidence Based Oligonucleotide) set on Nexterion® Slide E that they claim are compa-
                                              rable or superior to the array CGH equivalent currently offered by commercial vendors
Data was kindly provided by Dr. Norma         In Europe; the microarray core facility at UMCG Groningen has been successfully
Nowak, Roswell Park Cancer Institute          working with Nexterion® Slide E for aCGH.


                                              The examples show that four of the Nexterion® slide surfaces have been successfully
                                              used in array CGH. SCHOTT has worked with these facilities to produce a fully opti-
                                              mized protocol specifically for the array-CGH application on the Nexterion® coated
                                              slides.   Please   contact     us    if   you  would     like  further  information
                                              (coatedsubstrate@schott.com).




                                              1.   Kallioniemi A. et al. (1992) Science 258:818-821.
                                              2.   Solinas-Toldo S. et al. (1997) Genes Chromosomes Cancer 20:399-407.
                                              3.   Pinkel D. et al. (1998). Nat Genet. 20:207-211.
                                              4.   Snijders A. M. et al. (2001) Nat Genet. 29:263-264.
                                              5.   Pollack J. R. (1999) Nat Genetics 12:41-46.
                                              6.   Lucito R. et al. (2000) Genome Res. 10:1726-1736.
                                              7.   Lucito R. et al. (2003) Genome Res. 13:2291-2305.
                                              8.   Takeo S. et al. (2001) Cancel Genet Cytogenet. 130:127-132.
                                              9.   Cowell JK, Nowak NJ. (2003) Adv Cancer Res 91-125.
Microarray Solutions                                                                                                              page 6



          New Product Focus – Nexterion® LEB (Low Evaporation Buffer) Kit


                                                  An increasing number of microarray facilities are printing high density arrays to
                                                  increase the number of features per slide. Although higher density printing can be
                                                  performed using standard microarrayers, this can often result in significantly extended
                                                  rinting runs ranging from overnight to several days or even longer. Typically printing is
                                                  performed at 50% relative humidity, and significant evaporation of the aqueous printing
                                                  buffers from the source plates and printing pins can occur. This results in printing
                                                  aberrations and poor spot morphologies as well as a reduced number of spots printed
                                                  per filled pin.
                                                  Product Development
                                                  SCHOTT had many requests from around the world to develop a low evaporation
Fig.1 Example of spot morphology
                                                  printing buffer that is compatable with Nexterion® Slide E, SCHOTT’s market leading
possible with Nexterion® LEB-Buffer
                                                  epoxysilane coated slide. R&D at SCHOTT Microarray Solutions initiated a program to
                                                  develop a “low evaporation” printing buffer that would be suitable for use during long
                                                  print runs and would produce excellent spot morphology (Fig.1) without negatively
Facilities using Nexterion® LEB-                  affecting the overall performance of the microarrays.
Buffer:
                                                  Samples of the first LEB prototype printing buffer were sent out to several test sites in
 • Microarray Core Facility, Lund,                Europe and the U.S., and although the feedback from most sites was positive in terms
   Sweden                                         of reducing the effect of evaporation, the spot sizes varied enormously. Given such a
 • The Institute of Genomic                       variance in spot size, plus the stringent requirements of many customers for specific
   Research/The Pathogen                          spot sizes, it was decided that simply offering a single standard Low Evaporation Buffer
   Functional Genomics Resource                   would not meet the needs of the majority of customers.
   Center (TIGR / PFGRC)                          To circumvent this problem, and to allow maximum flexibility to the customers,
   Maryland, USA                                  SCHOTT came up with the concept of a LEB Kit consisting of an improved LEB buffer
   (see Article on Page 7)                        that does not contain any detergent, and a separate 1% Sarkosyl solution for optional
 • City of Hope Medical Center CA,                use by the customer. By varying the amount of Sarkosyl solution added to the LEB,
   USA                                            customers are able to adjust the spot size (50 µm–200 µm depending on pin diameter)
                                                  according to their requirements. Full guidelines are provided with the kit.
                                                  The LEB kit has been extensively tested internally, and by a group of beta test sites,
                                                  and, to our knowledge, the evaporation rate of the improved LEB is the lowest (<5%
Scatter Plot of Optimized Hybridization           loss at 50% relative humidity over 16 hour print runs) of any spotting buffer currently
    Hu.Thymus 555 Hu.Brain 647                    available on the market. With a pitch (spot spacing) of 160 µm, it is possible to achieve
                                                  25k spot density, without any wandering and/or coalescence of spots. It was found that
                                                  pre-spotting is an extremely important factor in obtaining the best possible results with
                                                  the LEB printing buffer, with 60-70 prespots after each source visit appearing to be
                                                  optimal.
                                                  City of Hope Medical Centre LEB Evaluation
                                                  At the City of Hope Medical Center, the need for a lower evaporation spotting buffer
                                                  coincided with the transition from spotting cDNA to microRNA oligonucleotides. When
                                                  the Ambion mirVANA miRNA oligonucleotide probe sets were printed in 3x SSC and
                                                  aqueous phosphate-based spotting buffers on Nexterion® Slide E, the facility recog-
                                                  nized that only a maximum of 20-25 slides could be consistently printed (with four spot
                                                  replicates per slide) due to the evaporation of probes on the pins during print runs. To
Fig. 2: Scatter plot of expression ratios         circumvent this problem, the Nexterion® LEB buffer plus varying concentrations of
(background subtracted) displayed from            detergent were tested with the microRNA oligonucleotides. After some initial experi-
optimized hybridization (Courtesy City of
Hope)
                                                  mentation, two customized LEB buffers containing 0.005% and 0.0075% detergent
                                                  were selected for their ability to consistently printing up to 100 consecutive slides, with
                                                  four spot replicates per slide. After further testing, it was found that for printing miRNA
                LEB UPDATE!                       probes onto Nexterion® Slide E and HiSens E slides, the LEB buffer containing 0.005%
SCHOTT is in the process of further improving     detergent gave the most consistanly reproducible results.
the LEB buffer to accommodate over 25k spots
per slide, further details will be included in    Optimal Use of Expensive Probes
future editions of Microarray Solutions. Read-    An unexpected benefit was that the City of Hope discovered that they could print up to
ers interested in receiving further information
on the tests conducted in-house and by our        500% more slides with the customized LEB than with the basic phosphate-based
external test centers should contact us           buffer. In addition, there was little or no evaporation from the 384-well plate during the
(coatedsubstrate@schott.com) to receive a         4-5 hours print runs. An example of a recent hybridization from a stringently controlled
copy of the “LEB Testing Report”.
                                                  print is shown in Figure 2.
Microarray Solutions                                                                                                    page 7



Technical Support in Focus – The Institute for Genomic Research (TIGR) and
              Nexterion® LEB (Low Evaporation Buffer) Kit

                                        The Institute of Genomic Research (TIGR) / The Pathogen Functional Genomics
                                        Resource Center (PFGRC) is a not-for-profit center dedicated to deciphering and
    Fig.1: Comparison of evaporation
                                        analyzing genomes. For years TIGR/PFGRC has been at the forefront of the genomics
              over 5 days
                                        revolution through ambitious scientific programs and has helped scientists around the
           (Courtesy of TIGR)
                                        world deepen the understanding of life by producing results in a diverse range of
                                        applications in the areas of medicine, agriculture, energy, the environment and
          Phosphate   Nexterion®        biodefense. Microarrays are one of the many powerful tools TIGR/PFGRC uses to
            Buffer    LEB Buffer        analyze thousands of genes simultaneously. In 2005, TIGR/PFGRC chose to partner
                                        with SCHOTT as its preferred microarray substrate provider. TIGR/PFGRC adopted
                                        Nexterion® Slide E as its substrate of choice after performing extensive tests
 DAY 1
                                        comparing different slides and surface chemistries from several substrate suppliers.
                                        Since then TIGR/PFGRC has been in close contact with the SCHOTT technical
                                        support specialists to obtain the best possible results with Slide E.

 DAY 2                                  TIGR/PFGRC has been successfully using SCHOTT’s Nexterion® Slide E for spotting
                                        their oligo and cDNA libraries for over a year. In recent discussions it became clear that
                                        TIGR/PFGRC would require a different print buffer to Nexterion® Spot, the standard
                                        phosphate based spotting solution offered by SCHOTT, as it moved to printing higher
 DAY 3                                  density arrays. In trying to print these higher density arrays with standard buffers, it
                                        was noticed that the buffer in the source plates was evaporating during the longer print
                                        runs. This required that the source plates be repeatedly refilled. TIGR/PFGRC was
                                        obviously keen to avoid this tedious, time consuming step and was also looking to
 DAY 4                                  make more efficient use of the probes by having a spotting solution available that could
                                        provide more spots per filled pin. After a significant amount of product development
                                        work, SCHOTT was able to provide TIGR/PFGRC with a prototype LEB buffer kit that
                                        appeared to give promising results in internal evaluations while sustaining the good
 DAY 5                                  features of the Nexterion® Spot print buffer in terms of uniform spot morphology and
                                        high signal intensities.

                                        TIGR/PFGRC received the first prototype LEB and, in close consultation with the
                                        SCHOTT technical support specialists and the R&D team in Germany, immediately set
                                        about evaluating the product for its higher density arrays. The feedback from
                                        TIGR/PFGRC was promising with the LEB proving to be successful in reducing the
                                        effects of evaporation. However, in a conference call with SCHOTT’s technical support
                                        team, TIGR/PFGRC expressed concern at the spot sizes (around 100 µm) with the
“We have a pretty valued relationship   LEB, and were keen to see smaller spot sizes (many other LEB test centers reported
with the scientists at SCHOTT.          that they were looking for a smaller or larger spot size with the LEB prototype, spot
They're always very attentive to our    sizes ranged from 70 µm to 140 µm depending on the customer’s set-up conditions).
needs and work closely with us to       Taking this feedback into account, SCHOTT prepared several different versions of the
ensure our production runs smoothly.”   LEB buffer with different amounts of detergent to produce varying spot sizes.

Kathy Ong, Research Associate at        TIGR/PFGRC ran several experiments with the buffers including evaporation tests (see
TIGR                                    Fig.1) and analysis of the spot morphology, signal intensity and spot size until they
                                        found the customized LEB most suitable for their arrays. LEB E2 performed the best in
                                        the evaluation, eliminating the problem of evaporation over the longer print run (little
                                        evaporation was seen even after 8 days of incubation at room temperature) and
                                        offering spot sizes averaging 65µm that were ideal for TIGR/PFGRC’s higher density
Ordering details for the new            arrays. Furthermore, after SCHOTT’s R&D team informed TIGR/PFGRC that 60-70
Nexterion® LEB kit                      pre-spots per filled pin were optimal for prespotting with the LEBs, TIGR/PFGRC found
                                        that 600 spots could be printed with one filled pin – four times the number of spots it
                                        managed to get with the standard aqueous buffers.
Item N°      Contents
1117902      • 100 ml LEB print         In conclusion, TIGR/PFGRC was extremely impressed with its interaction with
               buffer                   SCHOTT’s technical support specialists throughout the LEB evaluation and admitted
             • 10 ml 1% Sarkosyl        that SCHOTT’s overall commitment to technical support was a key factor when
               solution                 TIGR/PFGRC decided to switch slide suppliers. The LEB buffer selected by
             • Instructions for use     TIGR/PFGRC is now a key component in the microarray production process.
                                        TIGR/PFGRC is now able to successfully produce high density arrays with precise spot
                                        sizes and morphologies, high signal intensities and low evaporation rates.
Microarray Solutions                                                                                                     page 8


                                  SCHOTT Microarray Solutions News
                                         SCHOTT Microarray Solutions Unit Gains ISO 9001 Certification
                                         In June 2006, the Microarray Solutions unit successfully passed the DIN EN ISO
                                         9001:2000 certification.

                                         Availability of New Surface Chemistries in HiSens Range of Slides
                                         Following the launch of the Nexterion® HiSens range of slides earlier this year with the
                                         Nexterion® Slide A (Aminosilane) and Slide E (Epoxysilane) surface chemistries,
                                         SCHOTT is pleased to announce that HiSens slides will now be available with all sur-
                                         face chemistries in the Nexterion® product portfolio. This includes Nexterion® Slide A+
                                         (Aminosilane), Slide AL (Aldehyde) and Slide H (3-D hydrogel) surfaces.
                                         Demand for Nexterion® HiSens slides continues to be high, and comes on the back of
                                         exceptionally good feedback from beta test sites and first customers. Users are consis-
                                         tently obtaining up to an eight fold increase in signal intensity with HiSens slides over
Kai Griesenbrock, Head of Quality and    regular slides, and the product is proving invaluable for applications in which research-
Process Management, accepts the ISO      ers have only a limited amount of target material. The availability of HiSens slides in all
  9001 certificate from Dr. Michaela     surface chemistries means that HiSens slides can now be used for an even wider
Krenzer, Senior ISO auditor, SCHOTT      range of applications, such as protein microarrays.
                                         Please contact your local Sales Manager for further information.

                                         Nexterion® Web shop Success
                                         Do you prefer to do your shopping online? Are you tired of having to wait for purchase
                                         orders to be prepared and authorized before you can order products? Do you like the
                                         flexibility of being able to order products 24/7? Well, why not join the vast number of
                                         SCHOTT customers who regularly purchase products through the Nexterion® Web
                                         shop?
To visit our web shop, please go to:
                                         SCHOTT launched the Nexterion® Web shop in all regions in February 2004. Customer
US:                                      usage of the web shop has increased dramatically with the number of U.S. customers
www.us.schott.com/nexterion/shop         quadrupling in the past year alone. The web shop allows users access to information
                                         on the full, up-to-date SCHOTT portfolio of Nexterion® products, including pricing,
Rest of the world:                       simply by completing the short registration form. All orders placed on the web shop are
                                         then sent directly to our Sales Department for immediate processing enabling us to
www.schott.com/nexterion/shop
                                         have the products sent out to you without delay.
                                         SCHOTT re-launched the web shop in early 2006 by adding new products and infor-
                                         mation on the Nexterion® range and implementing a number of suggestions for im-
                                         provements from users. If you have encountered any problems with the Nexterion®
                                         Web shop, or have suggestions on how we can improve the web shop experience,
                                         please contact us at coatedsubstrate@schott.com.



                           Conference and Exhibition Calendar 2006/07

  Event                    Location                 Date                     Weblink
  Discovery-2-             Boston, USA              25 - 27 September        www.discovery2diagnostics.com/
  Diagnostics (D2D)                                 2006
  (Chips to Hits)
  Advances in Microarray   Amsterdam, Nether-       31 October - 02 No-      www.selectbiosciences.com/conferences/amt2006
  Technology               lands                    vember 2006
  ASCB Annual meeting      San Diego, USA           10 -13 December          www.ascb.org/meetings/am2006/index.cfm
                                                    2006
  LabAutomation 2007       Palm Springs, CA.        28 - 31 January 2007     www.labautomation.org/LA/LA07
                           USA
  Statusseminar Chip-      Frankfurt, Germany       01 - 02 February         www.dechema.de/chips
  technologie 2007                                  2007

                                          If you are interested in presentations given by SCHOTT at past conferences please
                                          contact us at coated.substrate@schott.com

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Microarray Solutions newsletter #2

  • 1. Microarray Solutions The Nexterion® Newsletter Edition # 2 / September 2006 ARRAY BASED COMPARATIVE GENOMIC HYBRIDIZATION – CURRENTLY THE HOTTEST TOPIC IN MICROARRAYING “Microarray Solutions” reports on the increasing popularity of array-CGH in the microarray community, and highlights how some of the most important facilities in North America are using Nexterion® slides for this application. …read more on pages 4 and 5 Also featured this month... page 3 INTERVIEW WITH JAMI LOMAX New Sales Manager for East Coast North America & Technical Support Specialist for North America page 6 NEW NEXTERION® LOW EVAPORATION BUFFER How the Nexterion® LEB Kit can solve the problem of source plate evapora- tion during extended print runs page 7 TECHNICAL SUPPORT IN FOCUS How SCHOTT technical support assisted The Institute for Genomic Research in the use of the LEB Kit with Nexterion® Slide E page 8 LATEST NEWS FROM SCHOTT Including ISO 9001 certification for the SCHOTT Microarray Solutions unit, new surface chemistries for the HiSens range of Slides, and the Nexterion® Web shop Microarray Solutions SCHOTT North America Inc. 5530 Shepherdsville Road Louisville, KY 40228 USA Phone: +1- 502-657-4417 Fax: +1- 502-966-4976 E-Mail: coatedsubstrate@us.schott.com www.us.schott.com/nexterion
  • 2. Microarray Solutions page 2 General Manager Talk Dear Reader, Welcome to the second edition of “Microarray Solutions”, the newsletter from SCHOTT. The response from readers around the world to the first edition of the newsletter was extremely positive, and I would like to thank all readers who provided us with feedback. We are pleased to welcome a new member to the SCHOTT Microarray Solutions team in the U.S., Jami Lomax (see interview on Page 3). Jami joined our company in June and will be the Sales Manager covering the Eastern region of North America. She will also assume the role of Technical Support Specialist for North America. Jami will no doubt be in contact with our North American customers very soon, and she will be supported in her Technical Support role by Dr. Rajendra Redkar from our R&D site in Duryea, PA. Both Jami and Raj will continue to provide the first class technical support that SCHOTT is known for in the microarray community. They will also both feature prominently alongside our R&D Director, Dr. Ruediger Dietrich, in the popular Techni- cal Support in Focus section of the newsletter (see Page 7). In Asia, SCHOTT has established an extensive distribution network to offer Nexterion® microarray products into this rapidly growing market. SCHOTT already had a strong relationship with several key microarray facilities in Asia, but in recent months we have received an increasing number of enquiries from the Far East. To meet these requests, SCHOTT will be making full use of its international network of sales offices, as well as working in partnership with experienced third party companies who distribute life sci- ence products in the Far East. Further details on the new sales infrastructure for SCHOTT in Asia will be announced very soon. This edition of the “Microarray Solutions” features an article on Array CGH, an increas- Dr. Lutz Wehmeier ingly popular application. The article gives an overview of this exciting application from General Manager our perspective, and that of some of our customers. It also demonstrates how a variety Nexterion – Microarray Solutions of surface chemistries offered by SCHOTT Microarray Solutions have been success- fully utilized for Array CGH. We also include a very interesting article on the new Nex- terion® Low Evaporation Buffer (LEB) kit that was recently launched by SCHOTT. The feedback we have had so far on this product from customers has been excellent, and our article on TIGR is a prime example of how the LEB Kit can be used to resolve a very common problem encountered by microarray users. I hope you enjoy this edition of Microarray Solutions, and thank you for choosing SCHOTT as your preferred microarray substrate supplier. Regards, Lutz Wehmeier
  • 3. Microarray Solutions page 3 SCHOTT Profile – Jami Lomax East Coast Sales Manager and Technical Support North America MS: How long have you worked for SCHOTT? And where did you work prior to SCHOTT? JL: I joined SCHOTT as Sales and Support Manager in early June this year. Prior to SCHOTT I worked with Spectrumedix LLC as an account manager and applications support consultant. I was previously a research technician for SCHOTT Nexterion R&D in Duryea, PA where I was involved in the development of the most of the current slides in our product portfolio. “Microarray Solutions” MS: What key skills are required to do your job? catches up with the most JL: I think one of the key skills necessary to be a sales manager and technical support recent addition to the consultant is organization, patience, and time management! Most importantly, how- SCHOTT Microarray Solu- ever, is the ability to quickly identify the needs of customers and be able to communi- tions team. cate with them clearly and concisely. MS: What do you do in technical support? JL: As technical support manager for North America, I help to troubleshoot any prob- lems customers have working with our products and also offer advice and protocol recommendations to customers prior to their slide evaluations. This can be done via e- mail or over the phone, but I am also able to go on-site to help work through protocols and provide tips on how to get the most from Nexterion® products. In my previous role working in R&D at SCHOTT, I played a key part in the development of several of the Nexterion® products, including protocol optimization, so I feel very comfortable offering advice to customers based on first-hand experience with the products. MS: Can you describe your typical work day? JL: The nice thing about this job is that every day offers new challenges and no two days are ever the same! On the sales side, I spend a lot of time travelling to visit cus- tomers throughout my territory, talking to new potential customers and servicing exist- ing customers. For technical support, a question from customers can obviously arrive at any time and usually requires an immediate response, usually involving a telephone conversation with the customer. MS: How do you respond to customer queries? JL: My response to customer queries usually depends on the type of customer and the Technical Support contacts: nature of the problem. The more basic questions can easily be addressed by either e- mail or telephone. Other queries require a more detailed response. For example, we North America usually offer customers the opportunity to discuss the SCHOTT products and protocols Contact: Jami Lomax in detail prior to their slide evaluation via a conference call. Or for customers who are E-mail: jami.lomax@us.schott.com experiencing significant problems getting a product to work for them, I may even spend some time in the lab with them to offer the best advice possible. Europe, Asia, Rest of the World MS: What are the most important aspects of technical support? Contact: Dr. Ruediger Dietrich JL: In my opinion, and fortunately that of SCHOTT, technical support is one of the E-mail: ruediger.dietrich@schott.com most valuable things a company can offer, particularly with regards to microarrays where there are so many variables that can ultimately have an effect on results. We pride ourselves on responding to customer problems as quickly as possible to minimize down time. SCHOTT has developed an excellent reputation for providing first-class customer support, and I will strive to maintain this. MS: What part of the job do you find most satisfying? JL: I think the most satisfying aspect is undoubtedly resolving a particularly problem- atic customer query and feeling the sense of relief and gratitude from the customer. Many people I deal with are under extreme pressure to deliver results and being able to support them and contribute in some way to their success is a nice aspect of the job, particularly when you see how passionate and dedicated they are to their research. MS: What's special about Nexterion®'s technical support? JL: When interviewed for the position at SCHOTT, one of the major things that struck me was SCHOTT’s commitment to technical support. Dr. Lutz Wehmeier, the General Manager, was adamant that one of SCHOTT’s key advantages over competitors was our comprehensive technical support services, and he felt that this, coupled with the product quality, was a significant reason for SCHOTT making such a big impact in the microarray substrate market in such a short time. The knowledge base and experience within SCHOTT is a big bonus. We have staff who have over eight years experience of working with the Nexterion® slide coatings and this allows us to produce very high quality slides as well as quickly troubleshoot customer problems. Our technical support team is also structured in such a way that we can offer an immediate response to cus- tomers wherever they are in the world.
  • 4. Microarray Solutions page 4 Customer Application Focus – Comparative Genomic Hybridization using Nexterion® Coated Slides SCHOTT Insert data / images • Array-CGH Background Technological advances in the field of genomics have resulted in the sequencing of genomes from several organisms, including the human genome. The availability of genomic data has made it possible to screen genetic abnormalities on the genome- wide scale. Comparative genomic hybridization (CGH) is a conventional molecular cytogenetic method capable of detecting deletions, additions and amplification of DNA copy numbers1. However, one of the limitations of this method is lower sensitivity or spatial resolution in detecting single copy deletion. Array-CGH, which is based on microarray technology, dramatically increases the resolution and dynamic range for detection of deletions and amplification. For instance, copy number changes at the level of 5-10 kilobases of DNA sequences can be detected using array-CGH as against 10 megabases using conventional CGH. In addition, array-CGH allows direct mapping Fig. 1 Results from Combimatrix of genetic aberrations on the chromosomes, does not require cell culture or metaphase Molecular Diagnostics of an Array- chromosome, and improves throughput of testing. As the array format lends itself to CGH experiment with Nexterion® automation, array-based CGH technology should soon be practical for diagnosis of HiSens A slides from SCHOTT. cancer or genetic diseases in the clinical setting. CMDX intends to offer these arrays for clinical diagnostic applications. Array-CGH consists of an orderly arrangement of human DNA probes derived from coding and non-coding regions on coated glass substrate2,3,4. The DNA can be pre- pared from BACs (Bacterial artificial chromosomes) amplified by PCR from cDNA li- braries or specific genomic sequences5. The probes are printed robotically on an ami- nosilane or other coated microarray slide2. Alternatively, Approaches such as genomic representation arrays6, oligonucleotide arrays7 and specific target arrays8 have been also successfully used. For typical array CGH analysis DNA from a test and normal reference sample are labelled with different fluorophores and co-hybridized to the ar- ray9. After hybridization the ratio of fluorescence intensity of test to reference DNA sample (log2 ratios) is calculated to measure the copy number changes. Samples that Fig. 2 High density human BAC 20K do not show any genetic aberrations will indicate a constant ratio across the genome array printed on Schott Nexterion® A+ area. Therefore, array-CGH offers a flexible platform for studying genetic abnormalities slides at genome wide level, which can be performed in a high throughput, and cost-effective way. Array-CGH in Clinical Applications Array-CGH has been used as a research tool that can address many human health problems that involves genetic abnormalities including carcinomas and specific condi- tions such as Down Syndrome. Recently, several commercial companies are convert- ing this research tool into a diagnostic product by offering array-CGH products and services for clinical laboratories. These companies are currently seeking clarification of regulatory requirements for array-CGH and are in process of getting FDA approval for clinical applications. In order to meet FDA regulations, data generated from array-CGH assays needs to meet certain criteria and this is the challenge, for example, facing many companies Data was kindly provided by Dr. Norma looking to offer arrays to the clinical diagnostics community. The data should show low Nowak, Roswell Park Cancer Institute levels of variability among the replicates, samples with identical copy numbers, and non-specific background. Therefore the protocol for producing array-CGH assays needs to be carefully optimized. Another key consideration in this process is the quality of coated microarray slide, as this is critical for optimal immobilization of the probes and their accessibility to the target for hybridization, as well as for obtaining a low hy- bridization background. The coated glass substrate not only influences these factors, but is also important in determining whether users can consistently generate repro- ducible results day in and day out. SCHOTT is one of the few slide manufacturers able to offer the high level of lot to lot reproducibility required for diagnostic type applica- tions.
  • 5. Microarray Solutions page 5 Nexterion® Slide A and Slide A+ for array-CGH Applications The most popular slide surface chemistry for array-CGH has traditionally been Ami- Fig. 3: Representative human 19K nosilane, with Nexterion® Slide A and Slide A+ having performed exceptionally well for BAC aCGH profile of melanoma tumor this type of application at numerous facilities throughout the world. The Microarray and demonstrating detection of DNA seg- Genomics Facility at Roswell Park Cancer Research Institute (RPCI) in Buffalo, NY is ment copy number changes. Average one of SCHOTT’s oldest North American customers, having successfully used both log2 ratios (y-axis) were plotted for all Slide A and Slide A+ for an array-CGH application with a slightly modified version of clones based on chromosomal posi- the recommended SCHOTT protocols. Currently they use Nexterion® Slide A+ slides to tion (x-axis) with the blue bar demar- print their high-density, and Nexterion® Slide A to print their lower density (<15k) BAC cating the chromosomes (top plot) and arrays. The arrayed products have excellent morphology and retention on both slide centromeres (bottom plots). Horizontal surfaces. Slide A+ allows RPCI to print over 70,000 features per slide thanks to the red lines indicate the log2 ratio for hydrophobic nature of the surface chemistry. SCHOTT’s state-of-the-art slide produc- each segment as segmented by Circu- tion facility and experienced production team in Germany, manufacture slides with a very high lot-to-lot consistency, helping to minimise variance between print runs, and lar Binary Segmentation (CBS). giving the RPCI facility the confidence to use these slides for research projects lasting several years. Figure 1: High density human BAC array printed on Schott Nexterion type A+ The protocol followed by Roswell is relatively simple, with minimal post-print process- slides. ing required for either of the Nexterion® Aminosilane slide surfaces after spotting BAC The figure represents an overlay pseudo- printing solutions. In the RPCI facility, the arrayed slides are stored in a desiccated color image of a RPCI-119 Chr 5 Chr BAC microar- 17 Chr environment after printing and UV cross linked immediately prior to hybridization. Suc- ray, co-hybridized with fluorescently cinic anhydride blocking, boiling and/or SDS washes did not improve the performance labeled DNA derived from a melanoma sample (Cy5™) and a 5 Chr pooled human of the slides, but in fact added background and other reagent related artefacts that can reference control (Cy3™). LM-PCR be detected after scanning. The success achieved in the RPCI facility using SCHOTT representations of 19,000 BAC DNAs Nexterion® slides has led to the decision to utilize SCHOTT Nexterion® slides exclu- were spotted in duplicate on Nexterion® sively to manufacture BAC arrays for Empire Genomics, a spin-off company from RPCI Slide A+ using a BioRobotics MicroGrid II TAS arrayer and MicroSpot 10K split pins recently launched by Dr. Norma Nowak. Dr. Nowak commented, “We are successfully (Genomic Solutions, Inc. Ann Arbor, MI) using SCHOTT Nexterion® slides in our BAC aCGH services division of Empire Ge- at 150 µm spacing. Competitive hybridi- nomics. This product enables us to analyze thousands of test DNAs and to reliably zation of the melanoma sample with the compare their copy number status to control samples. When it comes to printing thou- reference control Chr 9 copy number identifies amplifications (red spots) and deletions sands of BAC elements at high density over thousands of slides, Nexterion® Slide A+ (green spots). from SCHOTT is clearly the best slide out there.” (Fig. 3) Other SCHOTT slides successfully used for array-CGH In North America., SCHOTT has been receiving an increasing number of enquiries Chr 17 from the microarray community for Nexterion® coated substrates for array CGH. Com- bimatrix Molecular Diagnostics (Nexterion® Slide A) (Fig. 1), UCSF Comprehensive Cancer Center (Slide A, A+ and E) and BC Cancer Agency (Slide AL) have all been successful in using Nexterion® substrates for array CGH with BAC clones. Stanford University has produced arrays for CGH using Invitrogen’s HEEBO (Human Exonic Evidence Based Oligonucleotide) set on Nexterion® Slide E that they claim are compa- rable or superior to the array CGH equivalent currently offered by commercial vendors Data was kindly provided by Dr. Norma In Europe; the microarray core facility at UMCG Groningen has been successfully Nowak, Roswell Park Cancer Institute working with Nexterion® Slide E for aCGH. The examples show that four of the Nexterion® slide surfaces have been successfully used in array CGH. SCHOTT has worked with these facilities to produce a fully opti- mized protocol specifically for the array-CGH application on the Nexterion® coated slides. Please contact us if you would like further information (coatedsubstrate@schott.com). 1. Kallioniemi A. et al. (1992) Science 258:818-821. 2. Solinas-Toldo S. et al. (1997) Genes Chromosomes Cancer 20:399-407. 3. Pinkel D. et al. (1998). Nat Genet. 20:207-211. 4. Snijders A. M. et al. (2001) Nat Genet. 29:263-264. 5. Pollack J. R. (1999) Nat Genetics 12:41-46. 6. Lucito R. et al. (2000) Genome Res. 10:1726-1736. 7. Lucito R. et al. (2003) Genome Res. 13:2291-2305. 8. Takeo S. et al. (2001) Cancel Genet Cytogenet. 130:127-132. 9. Cowell JK, Nowak NJ. (2003) Adv Cancer Res 91-125.
  • 6. Microarray Solutions page 6 New Product Focus – Nexterion® LEB (Low Evaporation Buffer) Kit An increasing number of microarray facilities are printing high density arrays to increase the number of features per slide. Although higher density printing can be performed using standard microarrayers, this can often result in significantly extended rinting runs ranging from overnight to several days or even longer. Typically printing is performed at 50% relative humidity, and significant evaporation of the aqueous printing buffers from the source plates and printing pins can occur. This results in printing aberrations and poor spot morphologies as well as a reduced number of spots printed per filled pin. Product Development SCHOTT had many requests from around the world to develop a low evaporation Fig.1 Example of spot morphology printing buffer that is compatable with Nexterion® Slide E, SCHOTT’s market leading possible with Nexterion® LEB-Buffer epoxysilane coated slide. R&D at SCHOTT Microarray Solutions initiated a program to develop a “low evaporation” printing buffer that would be suitable for use during long print runs and would produce excellent spot morphology (Fig.1) without negatively Facilities using Nexterion® LEB- affecting the overall performance of the microarrays. Buffer: Samples of the first LEB prototype printing buffer were sent out to several test sites in • Microarray Core Facility, Lund, Europe and the U.S., and although the feedback from most sites was positive in terms Sweden of reducing the effect of evaporation, the spot sizes varied enormously. Given such a • The Institute of Genomic variance in spot size, plus the stringent requirements of many customers for specific Research/The Pathogen spot sizes, it was decided that simply offering a single standard Low Evaporation Buffer Functional Genomics Resource would not meet the needs of the majority of customers. Center (TIGR / PFGRC) To circumvent this problem, and to allow maximum flexibility to the customers, Maryland, USA SCHOTT came up with the concept of a LEB Kit consisting of an improved LEB buffer (see Article on Page 7) that does not contain any detergent, and a separate 1% Sarkosyl solution for optional • City of Hope Medical Center CA, use by the customer. By varying the amount of Sarkosyl solution added to the LEB, USA customers are able to adjust the spot size (50 µm–200 µm depending on pin diameter) according to their requirements. Full guidelines are provided with the kit. The LEB kit has been extensively tested internally, and by a group of beta test sites, and, to our knowledge, the evaporation rate of the improved LEB is the lowest (<5% Scatter Plot of Optimized Hybridization loss at 50% relative humidity over 16 hour print runs) of any spotting buffer currently Hu.Thymus 555 Hu.Brain 647 available on the market. With a pitch (spot spacing) of 160 µm, it is possible to achieve 25k spot density, without any wandering and/or coalescence of spots. It was found that pre-spotting is an extremely important factor in obtaining the best possible results with the LEB printing buffer, with 60-70 prespots after each source visit appearing to be optimal. City of Hope Medical Centre LEB Evaluation At the City of Hope Medical Center, the need for a lower evaporation spotting buffer coincided with the transition from spotting cDNA to microRNA oligonucleotides. When the Ambion mirVANA miRNA oligonucleotide probe sets were printed in 3x SSC and aqueous phosphate-based spotting buffers on Nexterion® Slide E, the facility recog- nized that only a maximum of 20-25 slides could be consistently printed (with four spot replicates per slide) due to the evaporation of probes on the pins during print runs. To Fig. 2: Scatter plot of expression ratios circumvent this problem, the Nexterion® LEB buffer plus varying concentrations of (background subtracted) displayed from detergent were tested with the microRNA oligonucleotides. After some initial experi- optimized hybridization (Courtesy City of Hope) mentation, two customized LEB buffers containing 0.005% and 0.0075% detergent were selected for their ability to consistently printing up to 100 consecutive slides, with four spot replicates per slide. After further testing, it was found that for printing miRNA LEB UPDATE! probes onto Nexterion® Slide E and HiSens E slides, the LEB buffer containing 0.005% SCHOTT is in the process of further improving detergent gave the most consistanly reproducible results. the LEB buffer to accommodate over 25k spots per slide, further details will be included in Optimal Use of Expensive Probes future editions of Microarray Solutions. Read- An unexpected benefit was that the City of Hope discovered that they could print up to ers interested in receiving further information on the tests conducted in-house and by our 500% more slides with the customized LEB than with the basic phosphate-based external test centers should contact us buffer. In addition, there was little or no evaporation from the 384-well plate during the (coatedsubstrate@schott.com) to receive a 4-5 hours print runs. An example of a recent hybridization from a stringently controlled copy of the “LEB Testing Report”. print is shown in Figure 2.
  • 7. Microarray Solutions page 7 Technical Support in Focus – The Institute for Genomic Research (TIGR) and Nexterion® LEB (Low Evaporation Buffer) Kit The Institute of Genomic Research (TIGR) / The Pathogen Functional Genomics Resource Center (PFGRC) is a not-for-profit center dedicated to deciphering and Fig.1: Comparison of evaporation analyzing genomes. For years TIGR/PFGRC has been at the forefront of the genomics over 5 days revolution through ambitious scientific programs and has helped scientists around the (Courtesy of TIGR) world deepen the understanding of life by producing results in a diverse range of applications in the areas of medicine, agriculture, energy, the environment and Phosphate Nexterion® biodefense. Microarrays are one of the many powerful tools TIGR/PFGRC uses to Buffer LEB Buffer analyze thousands of genes simultaneously. In 2005, TIGR/PFGRC chose to partner with SCHOTT as its preferred microarray substrate provider. TIGR/PFGRC adopted Nexterion® Slide E as its substrate of choice after performing extensive tests DAY 1 comparing different slides and surface chemistries from several substrate suppliers. Since then TIGR/PFGRC has been in close contact with the SCHOTT technical support specialists to obtain the best possible results with Slide E. DAY 2 TIGR/PFGRC has been successfully using SCHOTT’s Nexterion® Slide E for spotting their oligo and cDNA libraries for over a year. In recent discussions it became clear that TIGR/PFGRC would require a different print buffer to Nexterion® Spot, the standard phosphate based spotting solution offered by SCHOTT, as it moved to printing higher DAY 3 density arrays. In trying to print these higher density arrays with standard buffers, it was noticed that the buffer in the source plates was evaporating during the longer print runs. This required that the source plates be repeatedly refilled. TIGR/PFGRC was obviously keen to avoid this tedious, time consuming step and was also looking to DAY 4 make more efficient use of the probes by having a spotting solution available that could provide more spots per filled pin. After a significant amount of product development work, SCHOTT was able to provide TIGR/PFGRC with a prototype LEB buffer kit that appeared to give promising results in internal evaluations while sustaining the good DAY 5 features of the Nexterion® Spot print buffer in terms of uniform spot morphology and high signal intensities. TIGR/PFGRC received the first prototype LEB and, in close consultation with the SCHOTT technical support specialists and the R&D team in Germany, immediately set about evaluating the product for its higher density arrays. The feedback from TIGR/PFGRC was promising with the LEB proving to be successful in reducing the effects of evaporation. However, in a conference call with SCHOTT’s technical support team, TIGR/PFGRC expressed concern at the spot sizes (around 100 µm) with the “We have a pretty valued relationship LEB, and were keen to see smaller spot sizes (many other LEB test centers reported with the scientists at SCHOTT. that they were looking for a smaller or larger spot size with the LEB prototype, spot They're always very attentive to our sizes ranged from 70 µm to 140 µm depending on the customer’s set-up conditions). needs and work closely with us to Taking this feedback into account, SCHOTT prepared several different versions of the ensure our production runs smoothly.” LEB buffer with different amounts of detergent to produce varying spot sizes. Kathy Ong, Research Associate at TIGR/PFGRC ran several experiments with the buffers including evaporation tests (see TIGR Fig.1) and analysis of the spot morphology, signal intensity and spot size until they found the customized LEB most suitable for their arrays. LEB E2 performed the best in the evaluation, eliminating the problem of evaporation over the longer print run (little evaporation was seen even after 8 days of incubation at room temperature) and offering spot sizes averaging 65µm that were ideal for TIGR/PFGRC’s higher density Ordering details for the new arrays. Furthermore, after SCHOTT’s R&D team informed TIGR/PFGRC that 60-70 Nexterion® LEB kit pre-spots per filled pin were optimal for prespotting with the LEBs, TIGR/PFGRC found that 600 spots could be printed with one filled pin – four times the number of spots it managed to get with the standard aqueous buffers. Item N° Contents 1117902 • 100 ml LEB print In conclusion, TIGR/PFGRC was extremely impressed with its interaction with buffer SCHOTT’s technical support specialists throughout the LEB evaluation and admitted • 10 ml 1% Sarkosyl that SCHOTT’s overall commitment to technical support was a key factor when solution TIGR/PFGRC decided to switch slide suppliers. The LEB buffer selected by • Instructions for use TIGR/PFGRC is now a key component in the microarray production process. TIGR/PFGRC is now able to successfully produce high density arrays with precise spot sizes and morphologies, high signal intensities and low evaporation rates.
  • 8. Microarray Solutions page 8 SCHOTT Microarray Solutions News SCHOTT Microarray Solutions Unit Gains ISO 9001 Certification In June 2006, the Microarray Solutions unit successfully passed the DIN EN ISO 9001:2000 certification. Availability of New Surface Chemistries in HiSens Range of Slides Following the launch of the Nexterion® HiSens range of slides earlier this year with the Nexterion® Slide A (Aminosilane) and Slide E (Epoxysilane) surface chemistries, SCHOTT is pleased to announce that HiSens slides will now be available with all sur- face chemistries in the Nexterion® product portfolio. This includes Nexterion® Slide A+ (Aminosilane), Slide AL (Aldehyde) and Slide H (3-D hydrogel) surfaces. Demand for Nexterion® HiSens slides continues to be high, and comes on the back of exceptionally good feedback from beta test sites and first customers. Users are consis- tently obtaining up to an eight fold increase in signal intensity with HiSens slides over Kai Griesenbrock, Head of Quality and regular slides, and the product is proving invaluable for applications in which research- Process Management, accepts the ISO ers have only a limited amount of target material. The availability of HiSens slides in all 9001 certificate from Dr. Michaela surface chemistries means that HiSens slides can now be used for an even wider Krenzer, Senior ISO auditor, SCHOTT range of applications, such as protein microarrays. Please contact your local Sales Manager for further information. Nexterion® Web shop Success Do you prefer to do your shopping online? Are you tired of having to wait for purchase orders to be prepared and authorized before you can order products? Do you like the flexibility of being able to order products 24/7? Well, why not join the vast number of SCHOTT customers who regularly purchase products through the Nexterion® Web shop? To visit our web shop, please go to: SCHOTT launched the Nexterion® Web shop in all regions in February 2004. Customer US: usage of the web shop has increased dramatically with the number of U.S. customers www.us.schott.com/nexterion/shop quadrupling in the past year alone. The web shop allows users access to information on the full, up-to-date SCHOTT portfolio of Nexterion® products, including pricing, Rest of the world: simply by completing the short registration form. All orders placed on the web shop are then sent directly to our Sales Department for immediate processing enabling us to www.schott.com/nexterion/shop have the products sent out to you without delay. SCHOTT re-launched the web shop in early 2006 by adding new products and infor- mation on the Nexterion® range and implementing a number of suggestions for im- provements from users. If you have encountered any problems with the Nexterion® Web shop, or have suggestions on how we can improve the web shop experience, please contact us at coatedsubstrate@schott.com. Conference and Exhibition Calendar 2006/07 Event Location Date Weblink Discovery-2- Boston, USA 25 - 27 September www.discovery2diagnostics.com/ Diagnostics (D2D) 2006 (Chips to Hits) Advances in Microarray Amsterdam, Nether- 31 October - 02 No- www.selectbiosciences.com/conferences/amt2006 Technology lands vember 2006 ASCB Annual meeting San Diego, USA 10 -13 December www.ascb.org/meetings/am2006/index.cfm 2006 LabAutomation 2007 Palm Springs, CA. 28 - 31 January 2007 www.labautomation.org/LA/LA07 USA Statusseminar Chip- Frankfurt, Germany 01 - 02 February www.dechema.de/chips technologie 2007 2007 If you are interested in presentations given by SCHOTT at past conferences please contact us at coated.substrate@schott.com