Tecan Journal Edition 01/2009

Tecan Journal

Life Sciences ❙ Clinical Diagnostics ❙ Forensics ❙ OEM ❙ Business and Technical News

Edition 1 / 2009

Stepping up the pace for crop breeding projects pages 22-23

The power is in your hands with Freedom EVOware® page 5

Efficient drug research and development with the Freedom EVO® 75 pages 18 - 19

Getting under the skin of stem cells pages 24 - 25

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CEO WELCOME TECAN JOURNAL 1/2009

Welcome to an exciting New Year...

Welcome

...the Association for Laboratory Automation (ALA) exhibition is, as ever, a great way to welcome in the New Year. This venue is the perfect opportunity for us to show you how Tecan products continue to provide the innovation, value and flexibility demanded by your application requirements. Our newest products include the flexible MultiChannel Arm™ 384, improved labware logistics with nested disposable tips, new automated solutions for Tecan’s flagship microtiter plate reader Infinite® M1000, and the new Cavro® Omni Robot and Cavro® Centris Pump for OEM customers. We are also introducing advances in software innovation, with easy and simple Instant Pipetting™ and exciting Common Notification System (CNS) Technology enabling remote monitoring using your mobile devices, webpage, or Windows Vista® gadgets.

Vista is a registered trademark of Microsoft Corporation.

As well as telling you more about some of these products, this latest edition of the Tecan Journal shows how more and more of our customers’ challenges are being addressed and solved by Tecan technologies.

As ever, we invite you to talk to Tecan and see how you too can bring efficiency and effectiveness to your laboratory.

I would like to take this opportunity to sincerely thank you for the continued interest, support and trust you are placing in Tecan, our products, services and, most importantly, in our people. We will do everything to meet your expectations in the coming year 2009 and jointly strive for sustained mutual success.

We hope you enjoy this issue and, if you’re at ALA, come and see us at booth number 205.

Thomas Bachmann, CEO

CONTENTS TECAN JOURNAL 1/2009

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pg 7

The launch of the regional headquarters reflects the Company’s significant growth and strong commitment to the market in China pg 9

CEO welcome

4 Infinite® M1000 and Infinite® F500 enjoy series of drug discovery assay validations 5 The power is in your hands with Freedom EVOware® 5 Tecan launches new range of disposable tips for MultiChannel Arm™ 96 6 Tecan and GE Healthcare collaborate for high throughput process development 7 China and Asia Pacific operations expanded 8 Investing in success 8 Consolidating our organization for customers 9 Service laboratory benefits from Tecan’s support 10-11 Accuracy to the nth degree 12-13 Meeting the demands of everchanging liquid handling workflows

pg 24-25

New microarray-based tool is developed for identifying previously difficult to diagnose primary tumors in cancer patients

Contents

pg 18-19

14-15 Semi-automated process halves investigation time for protein-protein interactions 16-17 How stressed are you?

18-19 Efficient drug research and development with the Freedom EVO® 75 20-21 MicroRNA expression profiling of carcinomas of unknown origin 22-23 Automation accelerates crop improvement 24-25 Getting under the skin of stem cells 26-27 Automated processing for pioneering translational medicine project 28-29 Signaling success 30-31 Collaboration paves the way for flexibility in diagnostic laboratories 31 Get your views across with T@lk to Tecan 32

Events 2009

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PRODUCT NEWS TECAN JOURNAL 1/2009

Infinite® M1000 and Infinite® F500 enjoy series of drug discovery assay validations Tecan is a global leader in laboratory automation and detection, and its high-end Infinite multimode readers enjoy an enviable market position in terms of assay validation, for both premium Quad4 Monochromators™- and filter-based instruments.

Tecan is collaborating with providers of life science and drug discovery kits and reagents to validate its top-class multi-mode readers, the high-end, premium Quad4 Monochromators-based Infinite M1000, and Tecan’s fast and most sensitive filter-based detection platform yet, the Infinite F500, with a wide range of assay systems.

So far these include: LanthaScreen™, GeneBLAzer®, Adapta™ Universal Kinase, Z’-LYTE®, Omnia® PolarScreen™ and Predictor™. Both instruments are granted LanthaScreen Certified Plus status by Invitrogen Corporation.

LanthaScreen™ Certified+

The Infinite F500

Transcreener® Far Red FP - the Infinite M1000 and Infinite F500 are both Transcreener Far Red FP validated by BellBrook Labs, LLC.

HTRF® - both instruments have successfully passed the HTRF compatible validation program managed by Cisbio bioassays.

The Infinite M1000 is HTRF compatible in white plates.

DLReady™ - both instruments have met Promega®’s acceptance criteria for being certified as DLReady in the corresponding validation program managed by Promega.

This widespread validation reflects the high performance criteria of the Infinite systems across a wide range of detection modules and options, and provides choice for the customers whether to focus the work based on monochromator or filter-driven multimode systems.

To find out more on Tecan’s Infinite multimode readers, visit www.tecan.com/infinitem1000.

The Infinite M1000

LanthaScreen, Adapta, PolarScreen and Predictor are trademarks of Invitrogen Corporation. GeneBLAzer, Omnia and Z’-LYTE are registered trademarks of Invitrogen Corporation.

Transcreener is a registered trademark of BellBrook Labs, LLC, USA. DLReady is a trademark and Promega is a registered trademark of Promega Corporation, Madison, USA. HTRF is a registered trademark of Cisbio international, France.

PRODUCT NEWS TECAN JOURNAL 1/2009

The power is in your hands with Freedom EVOware® Tecan software continues to bring reliability and process security to users of automation.

Freedom EVOware 2.2 software makes it easier than ever to control pipetting and scheduling operations of Freedom EVO® liquid handling platforms. Based on the same intuitive graphical user interface, version 2.2 of this software offers greater flexibility and ensures full compatibility with the latest hardware modules – all without compromising on operating speed and process security.

With Instant Pipetting™, Tecan introduces a completely new way to control pipetting robots, without the user needing any previous knowledge of programming.

Instant Pipetting uses a touchscreen to enable users with any skill level to define applications and directly control pipetting with a liquid handling robot in real time.

This innovative feature allows the user to perform common pipetting operations, like sample distribution, reagent addition and serial dilution, in a very easy and interactive way. All steps can be repeated to perform multiple iterations, or saved and recalled as a

Software innovation Program

Define your pipetting step easy and simple

Instant Pipetting

RunControl

Recall your pipetting easily via iPhone

Phone RunControl

Monitor

Monitor your instrument while performing your pipetting step

CNS on iPhone or CNS gadget on desktop

Monitor your liquid channel and plates while performing your pipetting step

iPhone monitoring applications

script in Freedom EVOware or mobile phone applications, for increased ease and speed.

Once your assay is running, it is now easier than ever for you to stay connected to your application with the Common Notification System (CNS). This technology enables direct remote monitoring using your mobile phone device, your office desktop through a Windows

Vista® gadget, or via any networked computer with a web browser. The status of the system is immediately visible and if user interaction is required, it will be brought to your attention.

To find out more on Tecan’s Freedom EVOware, visit www.tecan.com/freedomevoware.

Vista is a registered trademark of Microsoft Corporation.

Increased throughput and flexibility with Tecan quality MultiChannel ArmTM 96 nested disposable tips Tecan’s new disposable tip products for use with the MultiChannel Arm 96 option for Freedom EVO® automated liquid handling workstations offer higher throughput through improved logistics possibilities.

Tips without filters can be nested in passive stacks of up to 800 tips per SBS position on the deck, or externally in Te-Stack™ modules to provide a capacity of 3,840 tips per Te-Stack column, maximizing walk-away time and improving throughput. The nested design also offers environmental and logistical benefits, including reduced storage space requirements by 60 % compared to non-nested tip formats, lower transport costs and less wastage by using less packaging materials.

The fully SBS-compatible racks have been designed for increased throughput and reliability by optimizing the flexibility of the workstation deck space. Systems fitted with Te-Stack modules can now use the same SBS-format columns for tip racks and microplates, and platforms set up for nonSBS disposable tips can easily be converted.

All MCA 96™ disposable tips are produced in a cleanroom and are certified to be free of DNA, DNAse/RNAse, pyrogens and endotoxins. Non-nested disposable tips are also available in a sterile format.

To find out more on Tecan’s disposable tips, visit www.tecan.com/consumables.

Disposable tips stored externally in a Te-Stack module

Disposable tips nested in stacks on the workstation deck

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PRODUCT NEWS TECAN JOURNAL 1/2009

Tecan and GE Healthcare collaborate for high throughput process development In drug development, screening for optimal conditions can be a tedious and timeconsuming process that can often become a bottleneck. Time-to-clinic and time-tomarket are key factors for the success of any drug development project, making efficient process development a crucial component of the overall drug development workflow.

GE Healthcare’s PreDictor 96-well plates on a Freedom EVO workstation

With recognized expertise in their respective fields of laboratory automation and protein purification, Tecan and GE Healthcare are working together to bring increased quality and consistency to high throughput process development. The collaboration between the two leading suppliers focuses on screening of protein purification conditions, using the winning combination of Tecan’s Freedom EVO® automated workstation with GE Healthcare’s PreDictor™ 96-well plates pre-filled with GE Healthcare BioProcess™ chromatography media.

The PreDictor 96-well plates have been developed to evaluate conditions for binding, wash and elution in a format that allows miniaturized, parallel processing, with the added benefits of minimal cross-contamination and reduced sample consumption. The good correlation of data with those generated by chromatography columns make the plates an excellent tool for the initial screening of process conditions.

PreDictor plates can be operated with both centrifugation and vacuum protocols, manually or in automated workflows. The throughput, precision and robustness of the Freedom EVO platform ensures reproducible results from GE Healthcare’s 96-well process development technology, providing efficient parallel screening performed in a multivariate fashion. Automated workflows also offer hands-off operation and eliminate human error, resulting in uncompromising experimental reproducibility and consistency among replicates. The combination of the technologies substantially shortens time spent on process development and reduces overall time-to-clinic, while allowing the investigation of a much larger experimental space, resulting in greater understanding of the process. The unique solution for the optimization of process conditions provides process developers with quality, efficiency and reliability in downstream process development.

To find out more on Tecan’s protein purification solutions, visit www.tecan.com/proteinscience.

PreDictor and BioProcess are trademarks of GE Healthcare Companies.

CORPORATE NEWS TECAN JOURNAL 1/2009

China and Asia Pacific operations expanded Tecan has opened its new Asia Pacific region headquarters, based in the heart of Shanghai’s pharmaceutical and life science center. The launch of the regional headquarters reflects the Company’s significant growth and strong commitment to the market in China, as well as the whole Asia Pacific region, and will improve service and support for customers and distribution partners alike.

The new office was opened by Thomas Bachmann, CEO of the Tecan Group, on the 28th October, 2008. The ceremony was attended by key customers from around the Asia Pacific region, major distributors, officials and representatives from key Chinese Provinces, the Swiss and Austrian Embassies in China and the Tecan Group. Guests were welcomed to the Shanghai office by the CEO and were treated to a traditional Chinese ceremonial inauguration of the facility, followed by a celebratory dinner.

Thomas Bachmann thanks Peter Grünig for building up Tecan (Shanghai) Trading Co., Ltd.

Mr Bachmann said: “We are delighted to be making this positive move into what is almost certainly one of the fastest growing regions in life sciences today. With a stronger presence and new facilities, we will be able to improve sales coverage, service and support to all of our customers and partners in the entire Asia Pacific region.”

Dinner with our chinese guests

Kirsti Tavast (Head of Market Unit Asia Pacific) and Shinya Dobashi (new General Manager Tecan Japan Co.) in front of the Asia Pacific map

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CORPORATE NEWS TECAN JOURNAL 1/2009

Investing in success Tecan is investing in focused training for regional service managers to help them continually improve the excellent service support that customers have come to expect. European service director Wilfried Bartz has been responsible for the initiative, and explained why he believes this training will help provide better customer care: “Service managers usually come from a technical background – good engineers with the extra talent that makes them stand out from the crowd. The aim of this training is to provide service managers with a foundation of core skills to help develop

Tecan service managers and other course participants with one of the MarketingAkademie Hamburg lecturers

their regional operations. The introduction of a course specifically for service managers at MarketingAkademie Hamburg represents the best training for our managers, and offers an ideal forum to explore how servicing challenges are handled in different industrial environments. We specifically chose not to have a course exclusively for Tecan personnel in order to encourage exchange of ideas, and focus outside of technical issues.”

“Providing comprehensive customer support requires more than just rapid maintenance and servicing of hardware.

Service engineers have to be able to assess customer needs and advise where potential changes in practice could boost efficiency or revenue. This ethos has to come from the service manager and be filtered down to the field service staff, so the benefits of this approach need to be understood on a management level. We also work closely with the sales team before an order is agreed to help provide solutions for the customer’s needs, according to the specific application, technical aspects and support required.

Service management is a versatile and demanding job, and investing in our regional managers helps them to provide a professional and efficient service to customers.”

Consolidating our organization for customers Over the past year, a small group of representatives from the quality, finance and other management teams within Tecan has been working hard to improve our internal organization and establish a more efficient, more productive Tecan Management System.

In recent years, with the growth of the Tecan Group, a number of different management tools have evolved as the size and operations of the company have

dictated. By consolidating our standard operating processes into one integrated and well-documented system, we have created a strong basis from which to continue building all organizational units and make constant improvements within Tecan. Our new management system will be maintained by a dedicated department for Operational Excellence that is directly overseen by a member of the Executive Committee and covers all our business organizations.

This new initiative has streamlined decision-making processes within Tecan, concentrating our efforts with a strong customer focus, and we believe this new structure will help us to respond faster, in a more flexible way, to our customers and the market in general. Thank you for your continued support.

SERVICES AND CONSUMABLES TECAN JOURNAL 1/2009

Service laboratory benefits from Tecan’s support

The Freedom EVO 100 has been integrated with a microplate heat sealer and is accessed by a robotic arm

The Cellular Screening Center (CSC) in the Institute for Genomics and Systems Biology (IGSB) at the University of Chicago provides a cell screening service for drug discovery and development in several local faculties. The reliability of Freedom EVO® workstations, supported by Tecan’s excellent customer service, copes easily with high throughput liquid handling for various research projects.

Sam Bettis, Technical Director at the CSC, explained: “We are a service laboratory for researchers here at University of Chicago, as well as at University of Illinois at Chicago and at Northwestern University, performing their high throughput analysis projects such as whole genome siRNA screening and small molecule screening.”

Starting with a clean sheet, Sam and his colleagues designed the laboratory and selected the automation robotics, including two Freedom EVO liquid handling

Sam Bettis, Technical Director, and Nicolette Pavlovics, Research Technologist, study the Freedom EVO 100 through its PC terminal

workstations. The first Freedom EVO 100 has an integrated liquid handling (LiHa) arm with eight independent tips, and is capable of constructing rearrays of RNAi master plates as well as dispensing into 96- and 384-well plates.

In addition, a Freedom EVO 200, equipped with a LiHa arm with eight tips, a robotic manipulator (RoMa) arm and a Te-MO™384, is used for plate set-up and replication.

Sam continued: “The modularity of Tecan platforms allowed us to upgrade the Freedom EVO 200 by integrating a microplate heat sealer, which now sits to the right of the instrument and is accessible with the RoMa arm. We are also in the process of integrating a laboratory information management system that will allow us to streamline siRNA storage and retrieval. It will automatically export Microsoft Excel® files to allow cherry picking with the Freedom EVO, which will be great.”

“I chose the Freedom EVO platforms because I had worked with Tecan instruments previously, and talking to many other people

The Freedom EVO workstations at the CSC are able to process 384-well plates

in the field confirmed that apart from having the best reliability and precision, Tecan offered excellent service. Our laboratory can’t afford any downtime, so services like after-care support and annual service contracts are very important.”

“We have a complex set-up, so Tecan has helped us with some software-related tasks such as integrating the sealer with the Freedom EVO 200, and for the software upgrades of the Freedom EVO 100 to ensure that communication with the robotic arm is maintained. Tecan always responds very quickly and we have a fantastic service engineer located here in Chicago. Everybody at Tecan from sales to service has been excellent but generally, once we had become familiar with the instruments, there have been few issues; it turns out that we’re much better at breaking the robots than the robots themselves are!” Sam concluded: “We are very happy with the Tecan workstations, and I have recommended them to several laboratories.”

Excel is a registered trademark of Microsoft Corporation.

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LIQUID HANDLING TECAN JOURNAL 1/2009

Accuracy to the nth degree

Researchers at Quanterix in Cambridge, Massachusetts, USA, are relying on a Freedom EVO® 150 liquid handling workstation to provide reliable and consistent pipetting in the development of a revolutionary new technology for single molecule detection.

An optical fiber bundle. The higher magnification view (inset) shows the uniform individual reaction vessels

Quanterix Corporation in Cambridge, Massachusetts, USA, was founded in 2007 to develop a novel platform for single molecule detection. The revolutionary SiMoA™ (Single Molecule Array) technology was originally developed at Tufts University, Massachusetts, by Quanterix scientific founder Dr David Walt, and is based on arrays of femtoliter-sized reaction vessels arranged uniformly on the tip of an optical fiber bundle. Each reaction vessel is isolated from neighboring vessels, and when incubated with dilute solutions, can be used to trap single molecules according to Poisson statistics. Fluorescent signals are detected via a custom instrument using automated image analysis software.

Initial applications for this innovative technology are focused on the detection of protein biomarkers. Quanterix has combined single molecule detection with the reagents used in immunoassays to achieve a 1,000-fold increase in sensitivity over standard ELISA. The company hopes that enhanced sensitivity will lead to real clinical benefits for patients. David Hanlon, Director of Strategic Marketing and Collaborations at Quanterix, explained: “This technology employs very similar principles

Fluorescent image of the optical fiber bundle. Illuminated spots represent single protein molecules

to traditional ELISA methods, but instead of detecting many molecules per well as with traditional analog systems, our technology isolates individual molecules in femtoliter volume reactions for a digital output. This approach offers a significant increase in sensitivity relative to standard assays, so we are looking into applications where the improved detection limits will provide real value to clinical diagnostics. There are many clinical opportunities for improved biomarker detection, including the detection of proteins implicated in oncology and cardiovascular disease, as well as markers useful for monitoring infectious agents and inflammatory response. One exciting prospect is the possibility of switching from detection of biomarkers in blood to less invasive body fluids, where the same marker proteins may be present at much lower concentrations. Using the sensitivity of the SiMoA platform, we are hoping to characterize the expression of many of these biomarkers in saliva or urine. We expect that our technology could be of particular value for screening programs, where earlier diagnosis of a condition could potentially have a much more favorable outcome for the patient.”

LIQUID HANDLING TECAN JOURNAL 1/2009

Todd Campbell, Senior Automation Engineer, continued: “We currently use the Freedom EVO platform as a research and development tool to optimize our assays, ensuring consistency throughout every assay we run from day to day. We use the automated system for both liquid handling as well as the manipulation of our consumable test strips. With the assay protocols under continuous evolution, this makes it much easier to make small adjustments to the system. The Freedom EVO offers the flexibility we need to make these changes as necessary, and the software is easy to use.

This has enabled us to develop our assay platform more quickly, and begin testing samples with our instrument.”

Jeffrey Randall, Principal Scientist, explained why Quanterix opted for Tecan systems: “Our proprietary assay and detection system is at least 1,000 times more sensitive and much more accurate than the traditional bulk ELISA assays, so careful control of pipetting volumes is important to us. The Freedom EVO platform

Todd Campbell holding an optical fiber bundle

offers very repeatable pipetting, and this has allowed us to ramp up our throughput without sacrificing the consistency of our results. We looked at other automated liquid handling systems on the market, but they did not meet our requirements.”

In his role as senior automation engineer, Todd has worked closely with Tecan to optimize the Freedom EVO platform for Quanterix’s needs. “I have been very impressed with Tecan’s support. When we were first setting up the system, our sales engineer was very helpful and extremely knowledgeable about the system. In the past, I have often found that equipment manufacturer’s sales personnel are not necessarily system specialists, but that is certainly not the case with Tecan. With our Freedom EVO systems in heavy use every day, it is especially important that any issues we have are resolved quickly.

Whenever we have needed support, they have been here within 24 hours, which is very impressive.”

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(l to r) David Rissin, Senior Scientist, Jeffrey Randall, Principal Scientist, and Todd Campbell, Senior Automation Engineer, with the Freedom EVO SiMoA is a trademark of Quanterix Corporation.

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APPLICATION BIOPHARMA TECAN JOURNAL 1/2009

Meeting the demands of ever-changing liquid handling workflows The Bioprocess, Analytical and Formulation Sciences Group at Merck, West Point, Pennsylvania, USA, has accumulated several Tecan instruments over the years for the development and automation of high throughput biological assays, essential in the support of bioprocess development of vaccines and biological products.

The Bioprocess, Analytical and Formulation Sciences Group at the Merck research and manufacturing facility at West Point, Pennsylvania, develops biological methods for a number of processes involved in the production of therapeutic proteins and vaccines. Over several years, the group has built up a wealth of Tecan liquid handling and detection systems and continues to develop new and innovative ways of automating its ever-changing workflow.

Kristine Little, Research Biochemist, said: “We use our Tecan systems to develop automated assays and provide a routine service in testing and processing to laboratories within the West Point site using these assays. We also carry out activity and binding assays for final products of the purification processes; biochemical testing including assays for residual DNA and concentrations of proteins and process residuals; assays for residual host cell proteins and binding assays such as EC50 or IC50; and ELISA assays.”

“We have accumulated our collection of 14 Tecan systems over the past decade,

including several Freedom EVO® platforms equipped with microplate readers and the Power Washer 384™ microplate washer for processing immunoassays, and some older Genesis RSP™ liquid handling platforms, also incorporating different add-on modules,” Kristine continued. “The decks on all the instruments are set up to give us maximum flexibility so we can run a variety of assays on any workstation. Some are grouped to run immunoassays, whereas others run the protein and DNA assays – the ‘mix and read’ type of simple one- or two-step assays that can easily be automated.

These usually involve adding dye and buffer mixture to the sample, followed by mixing and measurement of fluorescence intensity, giving sensitive, selective and quick quantification of nucleic acids and proteins.”

Amy Bowman, Research Biochemist, continued: “The Tecan systems dedicated to ‘mix and read’ DNA and protein assays are set up to run five or six plates per run unattended, and we have a similar throughput for immunoassays of six plates at a time. Only one analyst is needed to

Tecan’s Te-Chrom™ module, developed in close collaboration with Atoll GmbH, allows efficient automated processing of Atoll’s MediaScout RoboColumns on the Freedom EVO platform. When combined with the Te-Stack™ module, the Te-Chrom is able to collect fractions of volumes as low as 25 μl.

APPLICATION BIOPHARMA TECAN JOURNAL 1/2009 (l to r) Kristine Little and Amy Bowman with their back-to-back Tecan system

set up the Tecan instrument and then, depending on the assays, the run time varies from two to 14 hours. The automated systems are in full-time use and the six analysts responsible for running the processmonitoring laboratories will often re-set more than one instrument on the same day.”

“Throughput and flexibility are the main reasons we continue to choose Tecan and why we have recently added even more systems to our collection. Our most recent acquisition is a new back-to-back system, built and customized for us by the Tecan Integration Group (TIG) in North Carolina, and consisting of a Freedom EVO 200 platform on the front and a Freedom EVO 150 platform at the back. Together these platforms will run various ELISA assays as a carouselbased, high throughput system, handling as many as 1,440 analyses in 20 96-well plates, unattended over the course of 24 hours.”

“For us, flexibility in programming is just as important as in the hardware; samples come to us in a diverse range of types and formats, and we need to be able to handle different numbers and types of samples from one run to the next,” Amy added. To achieve this we have written and developed some Visual Basic® software in-house so we can create customized worklists for our sample runs. It is also important that Tecan offers the choice of so many different devices compatible with its liquid handling systems.

We use several modules incorporated into our Genesis RSP and Freedom EVO platforms, including numerous Te-MO™ multichannel pipetting arms, four MCA™ 96 multichannel arms and one MCA™ 384. The multichannel arms allow us to increase throughput and will enable us to make use of microfluidic devices – for performing small-scale ondeck microscale processes – in our future development of purification methods.

We recently purchased the first of these devices – a MediaScout® 96-array RoboColumn high throughput purification column system from Atoll – and have successfully integrated this into one of our Freedom EVO platforms. This will carry out chromatography analysis, specifically high throughput screening of chromatographic resins, which is the next step in our expansion plans for the whole set-up.”

Visual Basic is a registered trademark of Microsoft Corporation.

MediaScout is a registered trademark of Atoll GmbH.

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APPLICATION BIOPHARMA TECAN JOURNAL 1/2009

Semi-automated process halves investigation time for protein-protein interactions The Department of Molecular Pharmacology at the Marseille Cancer Research Center, France, has developed a semiautomated yeast two-hybrid system with the Te-MO™ 96 Multichannel Pipetting Option from Tecan, halving the time taken to obtain reproducible results from six to three days.

The state-of-the-art Marseille Cancer Research Center has developed a strong interest in the molecular biology of cancer.

The Center’s molecular pharmacology laboratory is involved in ongoing investigations into potential therapeutic targets, using yeast two-hybrid (Y2H) screening techniques for the discovery of protein biomarkers. The Y2H system is a well-established method for screening of protein-protein interactions (PPIs), relying on PPI-dependent activation of a reporter gene for identification of positive clones. Historically, false positive results have been the major drawback of Y2H techniques, requiring multiple replicates and protracted cleaning procedures to ensure reproducibility. In order to streamline this process, the molecular pharmacology laboratory has developed a semi-automated protocol for Y2H screening.

Patrick Lécine with the Freedom EVO

Comparison of the phenotypic assay results.

Phenotypic assays were performed either with the replica-cleaning (upper panel) or the liquid dilution protocol (lower panel) to compare their efficiency in detecting positive interactions using the four phenotypic assays

Frederique Lembo inserts a plate for the Te-MO 96

Patrick Lécine is responsible for all yeastbased screening at the Center, and explained why they chose to develop a semi-automated protocol: “Manual replica plating for Y2H analysis requires plating out an excess of yeast, far more than is necessary for phenotype determination, in order to guarantee reproducibility. To eliminate false positives, several cleaning steps with a replica block are needed to remove the excess yeast. These are timeconsuming, repetitive, tedious and prone to experimenter-dependent factors, such as the amount of pressure applied to the replica block and the number of replica cleaning steps required. We have automated the replica plating protocol using a Te-MO 96 Multichannel Pipetting Option from Tecan, which improves colony definition and distribution for unambiguous phenotype analysis, and allows us to use a simplified dilution protocol and less yeast culture per colony. Cross-contamination is avoided and reproducibility is improved significantly;

APPLICATION BIOPHARMA TECAN JOURNAL 1/2009

the multiple replica cleaning steps are no longer required but we get the same results and the Y2H phenotyping assays are much faster.”

Patrick continued: “Yeast cultures are created with plasmids encoding a transcription factor (Gal4) fused to bait sequences, and a number of reporter genes requiring Gal4 activation. These are transformed with different sequences from cDNA libraries, then grown in modified synthetic complete (SC) liquid medium deficient in leucine, tryptophan and histidine. Positive clones are selected and re-plated in 96-well format on solid SC medium for four to five days to confirm the phenotype, before transfer to liquid SC medium deficient in leucine and tryptophan. After three days growth cultures reach stationary phase, and concentration is determined across all wells based on OD600 absorbance measurements. From this point the procedure is automated using the Te-MO 96.

Cultures are gently

re-suspended and diluted, before plating cultures onto selective SC media for identification of positive clones of one of three phenotypes (His+, Ura+ and 5-FAOr).

In addition, 5 μl of undiluted culture is plated onto a nitrocellulose filter (YPD) plate for the β-galactosidase activity assay. Starting with one 96-well donor microplate, the resuspension, dilution and plating out of yeast cultures takes only six minutes. It has been relatively simple to automate the pipetting steps on the Te-MO 96, thanks to the easyto-use Gemini™ software, and our semiautomated protocol can be used for any Y2H screening application by varying the yeast starter culture or the phenotyping media in the plates.”

“The reproducible pipetting of the TeMO 96 has allowed us to adopt this new dilution-based replica plating process,” Patrick concluded, “making automation possible by eliminating the need for replica block cleaning of plates, halving the time

required to identify PPIs and minimizing the volumes of liquid and solid media required.

However, the current protocol is limited by the incubation time on solid media required to achieve equal yeast uptake with each tip of the manual multichannel pipette. In the future, we intend to put the Te-MO unit together with our Freedom EVO® 150 workstation in order to improve the reproducibility of pipetting for smaller colonies, allowing us to reduce incubation times. This will also allow us to automate OD600 measurements for liquid colonies, allowing absorbance to be measured and dilutions to be normalized after only 24 to 36 hours of incubation. Together, these improvements should both reduce the total time required for phenotype analysis and give rise to a fully automated process.

The team at Tecan has been good to work with, and someone is always available for help and advice, so we hope to achieve full automation soon.”

Phenotypic assays obtained with liquid dilution of the controls. Phenotypic assays were performed on SC-WHL + 25 mM 3-AT and SC-WUL plates using the seven controls and a serial dilution starting from a OD600=2 to 0.001.

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LIQUID HANDLING TECAN JOURNAL 1/2009

How stressed are you?

Psychologists at Anglia Ruskin University, UK, are using a Tecan liquid handling platform to automate ELISA-based analysis of cortisol and IgA levels in their investigations into the relationship between different types of stress and the immune response.

Researchers in the Department of Psychology at Anglia Ruskin University, UK, are exploring the relationship between stress and responses of the human body, particularly the immune system, using specific examples to look at the differences between chronic and acute stress. Dr Matt Bristow, Senior Lecturer in Biological Psychology, explained: “We are concentrating on various groups of individuals, effectively trying to model stress in a controlled environment and looking closely at the immune responses of those individuals. For example, one of the most recent projects was a collaborative study into the experiences of carers – usually husbands, wives or partners – of patients with frontal temple dementia, a type of dementia similar to Alzheimer’s disease but with an earlier average age of onset of 55 years. Our initial study found that carers who reported longterm chronic stress tended to have higher levels of immunoglobulin A (IgA) rather than the lower levels we expected. These surprising findings may indicate that the immune systems of carers are reacting to the challenges of their day with large increases, or alternatively that IgA levels

are not suppressed by chronic stress as was previously thought. This on-going research reveals the potential for salivary biomarkers to help us understand the health implications of real-life stressful situations.”

“At the other extreme, we followed a number of volunteers through their first tandem parachute jump, and mapped the response of the immune system to this kind of acute stress. There were progressively huge increases in cortisol in their saliva at each step, starting with the day before the event, arriving at the parachuting centre, going onto the aircraft and lastly, the actual jump from the aircraft at 10,000 feet. A large, transient increase in their immune responses, measured by levels of IgA, the most abundant antibody type in saliva, follows the rise in cortisol. This is consistent with previous literature; in response to a brief challenging event such as acute stress, the immune system reacts by releasing natural killer cells, IgA and proteins. The levels of these drop off extremely quickly, with IgA levels returning to normal within 40 minutes, but cortisol is still high hours after the jump.”

LIQUID HANDLING TECAN JOURNAL 1/2009

“In another study, we discovered that, in a 10-day period, stressed undergraduates tend to have very volatile IgA levels, whereas their non-stressed counterparts have steady IgA levels, giving a flat profile. It is clear from this and other examples, that studying an immune profile over several days gives a much better indicator of stress levels compared to taking just a single measurement. We need to have a thorough understanding of variability in immune responses and therefore collect saliva samples several times a day for up to two weeks, creating thousands of samples that would be impossible to handle manually.”

Matt continued: “We have used a Tecan platform to automate our ELISAs since 2006 and our daily throughput has increased considerably, from 70 to 100 samples manually to over 600 on the Tecan

system. We have purposefully restricted ourselves to handling only saliva samples, as they are so much easier to collect than blood, and indeed can be collected by participants themselves, with far fewer worries about storage and health and safety. There are also now so many more biomarkers of interest detectable in saliva, including antibodies, DHEA, testosterone and C-reactive protein, a marker in heart disease. The system includes a PosID™ scanner for barcode recognition and AIS LIMS software for tracking, integration with databases and data printing so, once the samples are loaded and the assays start, we can just leave it running. We are required by Human Tissue Act legalization to track all human tissue samples through the laboratory and barcoding gives us complete certainty that no sample mix-up or mislabeling has occurred. In all, the system

eliminates human errors, schedules assays dynamically and very effectively to increase throughput, and gives us much more reliable data than before, in a far shorter time. I am very pleased that we managed to get a fully automated system that has delivered considerably more than our initial expectations.”

“The Tecan platform has given us extra capabilities and we are not yet running it at maximum capacity,” Matt concluded.

“We have already built up several academic collaborations with other research teams and units using the equipment. This consultancy service brings in more capital for the university and our department, allowing us to continue our research without worrying too much about funding, and the high capacity of the system means we still have room to deal with any studies that come our way.”

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APPLICATION BIOPHARMA TECAN JOURNAL 1/2009

Efficient drug research and development with the Freedom EVO® 75 Toa Eiyo in Japan is a pharmaceutical company specializing in the research and development of pharmaceutically unique medicinal products for cardiovascular diseases such as angina or heart attacks.

The Company’s pharmacokinetic testing laboratory in Fukushima has chosen the Freedom EVO 75 equipped with an 8 Plus 1 Access™ Liquid Handling Arm to achieve greater efficiency by automating its sample preparation steps.

The development of new drugs at Toa Eiyo starts with the synthesis and evaluation of various novel low molecular weight compounds at its Tokyo Research Laboratories. The compounds that demonstrate potency are selected for pharmacokinetic testing at Toa Eiyo’s Fukushima Research Laboratories. Mr Yoshiaki Watanabe, chief scientist, explained: “Preliminary pharmacokinetic studies using cells of human origin or artificial membranes are performed at our Tokyo Research Laboratories, and promising compounds are transferred to our section for further pharmacokinetic, toxicity and safety testing. Our section has around 20 members, of whom six are involved in pharmacokinetic studies, involving measurement of drug concentration in blood plasma from animal testing and clinical trials. In addition, Toa Eiyo’s Pharmaceutical Technology Center is making special efforts to develop drug formulations that are pharmaceutically unique, and pharmacokinetic profiles of

these new formulations are also evaluated in our section. With this recent increase in the number of samples we analyze, we wanted to automate the sample preparation steps to achieve greater efficiency. Tecan’s liquid handling devices greatly attracted our interest at the 8th International ISSX Meeting in October 2007, and we selected the Freedom EVO 75 with integrated 8 Plus 1 Access Liquid Handling Arm, which offered the management of pipetting precision with its liquid level detection system.”

They use the Tecan instruments for automated preparation of blood plasma samples, which involves solid phase extraction (SPE) or deproteinization in 96well filter plates, using customized programs for each step. As pipetting precision is critical, an integrated balance is used before each run to confirm the pipetting precision of the first channel, which is used for sample transfers, while all eight channels are used for other preparation tasks. Mr Watanabe

Toa Eiyo’s Pharmaceutical Technology Center develops pharmaceutically unique formulations for the treatment of heart disease.

Photo provided courtesy of Toa Eiyo

Toa Eiyo’s pharmacokinetic team (l to r, back row): Mr Yoshiaki Watanabe, Mr Kazuhiro Taniyama, Mr Takashi Sato (l to r, front row) Ms Sakiko Suzuki, Ms Marie Tanaka, Mr Toshihisa Kodama

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MICROARRAY PRODUCTS TECAN JOURNAL 1/2009

MicroRNA expression profiling of carcinomas of unknown origin

Loading samples into the HS 4800 Pro hybridization station in the service department at Exiqon

Around 5 % of all newly diagnosed metastatic cancers are carcinoma of unknown primary (CUP) origin, where the site of the primary tumor cannot be determined, despite the use of advanced immunohistochemical or radiological techniques.

Because effective cancer treatment depends on early identification of the primary tumor, CUP patients have poor prognoses, with a median survival of 3-6 months and a oneyear survival rate of less than 25 %.

Exiqon has developed a new molecular tool for the diagnosis of CUP, using microarraybased microRNA profiling data. MicroRNAs are good candidates for such a tool as their expression signatures can be used to classify specific cancers. Simultaneous detection of all microRNAs on an array presents a number of challenges. The short length (21-23 nt) of microRNAs leaves little room for probe optimization, and many microRNAs are very similar in sequence, varying by only one or two bases. Also, microRNAs possess huge variation in base composition, which results in a large TM range of microRNADNA probe dimers (Figure 1, gray bars). To overcome these challenges, the capture probes used in the miRCURY™ LNA™

microRNA Arrays incorporate Exiqon’s high affinity Locked Nucleic Acid (LNA) technology. The TM-normalized probes (Figure 1, yellow bars) of these arrays result in unparalleled sensitivity and excellent mismatch discrimination for all microRNAs, making them superior to DNA probes (Figure 2) for efficient discrimination between closely related microRNA family members and microRNA profiling from as low as 30 ng total RNA. Automation is an important factor for standardization of complex experimental procedures, such as those involved in hybridization experiments, to ensure reproducible and reliable data. Tecan’s HS Pro

series of hybridization stations – available as HS 400™ Pro, which handles up to four slides, and a larger version, HS 4800™ Pro, which can process up to 48 slides – enables full automation of microarray hybridization experiments with minimal handling of solutions and slides. The hybridization station applies washing buffer to the active slide surface within a hybridization chamber that seals the slide from the top to improve washing efficiency. During hybridization, the sample is subjected to the patented agitation mechanism to guarantee uniform incubation. The agitation, together with hybridization station’s unique active bubble suppression (ABS™) system, helps to achieve maximum specificity, sensitivity and reproducibility of results. The on-slide nitrogen drying procedure (OSND™) results in a low and uniform background, and the slides can be scanned immediately without the need for tedious drying procedures.

Exiqon developed the miRCURY LNA microRNA Arrays using the HS 4800 Pro, and three are in its R&D laboratories for product development. Another seven HS 4800 Pros are being used in the service department and are operated to full capacity, every day, where samples received from customers are processed. Although Exiqon’s arrays can be hybridized manually, the Company has always recommended automated processing using Tecan’s hybridization stations for the most reliable results.

To develop a classifier tool for diagnosis of CUP, Exiqon’s miRCURY LNA microRNA Arrays were processed on Tecan HS 4800 Pro hybridization station, for microRNA profiling of normal and tumor tissues. More than 500 tumor and normal adjacent tissue samples were collected from both fresh frozen and formalin fixed, paraffin embedded sections of 18 tissue types (adrenal, bladder, breast, cervix, colon, esophagus, gall bladder, kidney, liver, lung, ovary, pancreas, prostate, rectum, small intestine, stomach, testis and uterus), representing the most common tissues of origin for CUP. Total RNA from tissue was analyzed for microRNA expression on the

MICROARRAY PRODUCTS TECAN JOURNAL 1/2009

Figure 1. TM-normalized LNA capture probes. DNA capture probes (gray bars) for human microRNAs have a TM range of more than 30 °C and an average TM of 64 °C (StDev = 5 °C). The LNA capture probes (yellow bars) have a TM range of only 10.0 °C and an average TM of 71.5 °C (StDev = 1.6 °C).

Figure 2. LNA-based arrays are superior to DNAbased arrays at detecting microRNA. Upper panel: Exiqon’s LNA-based array. Lower panel: A DNA-based competitor array. Gray bars indicate the percentage of capture probes capable of detecting 50 amol of synthetic microRNA within the given range of GC content. Yellow bars indicate the percentage of capture probes not detected at 50 amol. It is clear that DNA-based arrays show decreasing efficiency with decreasing GC content.

miRCURY LNA microRNA Array using the HS 4800 Pro hybridization station, and a unique microRNA profile for each of the 18 tissue types was generated (the full protocol for hybridization and washing can be downloaded at www.tecan.com or at www.exiqon.com). A microRNA expression database was established, and a detailed analysis of these results revealed that a cancer classifier based on the expression levels of seven microRNAs is sufficient to distinguish between tumors from the 18 tissue types.

The classifier tool was subsequently used to identify the tumor type of a patient suffering from CUP by analyzing a biopsy from a lymph node metastasis. A biopsy from a lymph node metastasis from a patient suffering from CUP was profiled on the miRCURY LNA microRNA Array. The microRNA expression level of the metastasis was compared to the average expression level across the 18 tissues and, based on a simple cluster analysis, the lymph node metastasis microRNA profile was shown to

belong to the colorectal microRNA cluster (Figure 3). A primary colon tumor was later found in this patient, which highlighted the potential of this method as a tool for identifying primary tumors in CUP patients.

Therefore, in this case, one would – based on the microRNA profile alone – be able to direct the treatment against a colon cancer without having to perform a full body PET scan to search for the primary tumor. A colonoscopy would have been sufficient to verify this result.

Taken together, the results indicated that the miRCURY LNA Array used in conjunction with the HS 4800 Pro hybridization station is a very powerful combination for high quality microRNA expression studies. It permits highly specific probe binding by yielding maximum sensitivity and a high inter- and intra-slide reproducibility by circumventing the handling drawbacks and the decrease in data quality associated with manual hybridization experiments.

LNA and miRCURY are trademarks of Exiqon A/S.

Figure 3. The workflow and result of the identification of the origin of a primary cancer. The cluster analysis identified the lymph node metastasis microRNA profile as colorectal cancer. All the colorectal samples, including the metastasis, cluster together and clearly indicate that the colon was the origin of the metastasis.

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LIQUID HANDLING TECAN JOURNAL 1/2009

Automation accelerates crop improvement Scientists at the Institute of Plant Breeding of the University of Kiel, Germany, are developing TILLING® (Targeting Induced Local Lesions in Genomes) programs in rape seed and sugar beet, relying on their Freedom EVO® liquid handling workstation for high throughput DNA purification and normalization using MACHEREY-NAGEL NucleoSpin® 96 Plant DNA extraction kits.

The Institute of Plant Breeding, part of the Institute of Agronomy and Crop Science at the University of Kiel, is a research and teaching facility, conducting various plant breeding projects in crop species like sugar beet, barley, rape seeds and asparagus.

Some of the projects are funded by the German national genome program, Genome Analysis of the Plant Biological System (GABI), including rape seed and sugar beet TILLING projects. Tecan’s Freedom EVO 200 liquid handling workstation is being used for automated DNA extraction and normalization for establishing the TILLING projects in which, typically, there are 4,000 or more plants in a population from which DNA needs to be individually extracted.

Prof Christian Jung and Mrs Gislind Bräcker

“We chose Tecan’s workstation because we needed an automated, high throughput system to extract DNA,” explained Professor Christian Jung, Director of the Institute of Plant Breeding.

“Our Freedom EVO platform has an integrated centrifuge, so it is able to perform DNA purification using MACHEREY-NAGEL’s NucleoSpin 96 Plant kit, automating the whole procedure after homogenization of leaf material with a SPEX SamplePrep 2000 Geno/Grinder®, which uses small steel beads to process simultaneously up to 48 samples in 2 ml reaction tubes, or 192 samples using 8-strip microtubes in a 96-well format. The Freedom EVO also

normalizes the purified DNA, using the integrated GENios™ fluorescence reader to measure the DNA concentration in 96-well microplates and dilute the DNA in each well to the right concentration for PCR amplification. This is a huge help for our projects, because it is very tedious to normalize by hand, and almost impossible in micro plates, so the automation makes life much easier for the technician faced with normalizing thousands of DNA samples. The DNA needs to be of good quality, because it will be stored and used in PCR reactions for the next 10 to 20 years. DNA extracted manually may not be of sufficient quality to withstand long-term storage whereas, in combination with the MACHEREY-NAGEL extraction kits, we can obtain highly purified DNA that we can store for long periods. The greatest advantage of this system is that the entire purification and normalization procedures are automated, and this offers the technician time to perform other tasks.”

The institute has had the Freedom EVO since 2006, and Tecan and MACHEREY-NAGEL worked together to adapt and optimize the protocol for automated DNA extraction and normalization. Prof Jung added: “We have been using it routinely for about a year now, and our technician, Gislind Bräcker, is happy with its performance. The yield of DNA has

LIQUID HANDLING TECAN JOURNAL 1/2009

(l) The Institute of Plant Breeding at the University of Kiel (r) The Freedom EVO platform is used for high throughput DNA purification and normalization

been improved, and we can consistently purify several micrograms of DNA from each sample, processing up to 192 samples in 2 ml reaction tubes in a day. The complete DNA extraction process takes about 6.5 hours, including the four hours spent to prepare the starting material by homogenization of leaf tissue, removal of the steel beads, and a two-hour incubation and centrifugation.

In the future, we are planning to switch to using 8-strip microtubes in 96-well format, with which we expect the procedure to be quicker. The PCR set-up will also be automated on the Freedom EVO in the future, to further streamline our workflow.

For DNA normalization, 384 samples can be measured and adjusted in one day.”

TILLING involves the identification of mutants within specific genomic regions, among huge populations that have been mutagenized by ethylmethane sulfonate (EMS). M1 generation plants are mutagenized and self-pollinated to produce the M2 population, and the DNA from the leaves of each individual M2 plant is extracted and stored, while the plants are self-pollinated again and M3 generation seeds are produced. The DNA is analyzed by PCR in specific genomic regions of interest, and the amplified fragments are treated with endonuclease CelI, which

cleaves mis-matches, yielding additional fragments when there is a base substitution in the selected region. When the mutated sequence indicates the possibility of an altered phenotype, the corresponding batch of M3 seeds is analyzed, and the plants carrying the mutation are isolated for further study. “Plant breeders need new genetic variation and, because of the unpopularity of transgenic technology in Europe, mutation technology and TILLING is a new source for genetic variation. Breeders, particularly in Europe, are very interested in this technology. Identifying sequence variants or mutations in this way, by genotype rather than by phenotype, is now possible because more and more sequence information of crop species is available.

That is the reason why a number of these TILLING platforms have been established in different countries around the world for various crop species.”

Prof Jung continued: “We are currently developing TILLING platforms for rape seed and sugar beet, in collaboration with commercial plant breeders. For one project in particular, we are looking at a deleterious component in rape seed, called sinapin, that makes it unsuitable as animal fodder. We are looking for plants that have a mutation in one of the genes for sinapin

metabolism, and we expect that if one of these major genes is mutated, the plant will have a reduced amount of sinapin, and this mutant would then be suitable for feeding to animals. This is a clear practical breeding purpose, where the yield of our project can be used by commercial plant breeders.”

“Very recently, the animal breeding unit of the university has started to use the Freedom EVO for automated DNA extraction from cows for a program on mapping, genotyping and haplotyping cattle DNA. Although between us we have huge numbers of DNA extractions to perform, the Freedom EVO has the capacity to cope with these sample numbers,” concluded Prof Jung.

Geno/Grinder is a registered trademark of SPEX SamplePrep, Inc. NucleoSpin is a registered trademark of MACHEREYNAGEL, GmbH & Co. TILLING is a trademark of Arcadia Biosciences, Inc.

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DETECTION TECAN JOURNAL 1/2009

Getting under the skin of stem cells Researchers in the Center for Biosciences at the Karolinska Institute in Stockholm, Sweden, have been using an Infinite® 200 microplate reader and NanoQuant Plate™ for the characterization of a newly isolated population of skin stem cells in mice.

Researchers in the Center for Biosciences at the Karolinska Institute, Sweden, have recently isolated skin stem cells displaying several exciting properties which challenge previous assumptions. The study, published in Nature Genetics*, demonstrated that these stem cells (Lgr5+ cells) are the most primitive skin stem cells yet described, able to divide actively and migrate through the skin tissue during follicle growth and regression. These cells are partly regulated by the well characterized ‘Hedgehog’ signaling pathway, and are of particular interest to the wound-healing process and the development of basal cell carcinomas (BCC), the most prevalent form of skin cancer.

Characterization of gene expression in these cells has been particularly difficult due to their scarcity. Dr Maria Kasper, from the Karolinska Institute’s environmental toxicology group, explained: “These cells are extremely rare, and so the experimental material we obtain is especially valuable; 100,000 sorted cells only renders 150 ng of RNA. Once this material has been isolated, it is used to identify signaling pathways involved in stem cell regulation by qPCR.

The significance and reliability of the data obtained is dependent on both the quantity and integrity of the extracted RNA, as qPCR using very low starting concentrations of RNA is particularly sensitive to these variables.

Therefore, it is very important for us to achieve exact quantification of extracted samples with minimal loss of material.”

Traditional photometry-based systems for evaluating RNA samples are dependent on comparing absorbance measurements at multiple wavelengths, but most readers cannot produce reliable absorbance readings from small volumes of RNA sample.

However, using Tecan’s NanoQuant Plate in conjunction with a multi-functional Infinite 200 microplate reader offers the necessary sensitivity for reliable low volume RNA purity analysis. Maria commented: “Using this system ensures that only minimal material is required for reliable evaluation of our valuable RNA samples and allows us to investigate more than 30 selected genes by qPCR from the initial 150 ng of isolated RNA.”

Lgr5 expression in the mouse hair follicle.

Immunohistochemical staining showing localization of Lgr5 (blue) and CD34 (brown) expressing cells.

DETECTION TECAN JOURNAL 1/2009

The NanoQuant Plate is a 16-channel quartz optic for measurement of low concentrations and low sample volumes, with quick and simple washing procedures to eliminate cross-contamination.

Specifically designed to give outstanding performance and a very high rate of reproducibility, the NanoQuant Plate is intended for a broad range of applications requiring sample volumes as low as 2 µl.

Combined with the Infinite 200 microplate reader, this system allows accurate absorbance measurement of DNA/RNA concentrations as low as 1ng/μl, helping to conserve valuable samples.

Maria Kasper, Takashi Shimokawa and Viljar Jaks

Maria’s colleague, Dr Viljar Jaks, continued: “Having the NanoQuant Plate in the laboratory has been very useful for our investigations. Because it is integrated into the Infinite microplate reader, it is very easy to use and the i-control™ software is intuitive. Although our studies are not high throughput by nature, the 16-channel plate format allows us to analyze entire experimental sets together.”

“Accurate quantitation allows us to normalize RNA concentrations very precisely for further analysis of gene expression using a variety of methods, helping to identify the pathways contributing to the behavior of these stem cells. Elucidation of these regulatory mechanisms could help us to understand the role of keratinocyte stem cells in various forms of tissue growth.

One example of this is the wound healing process. Because hair follicles are laid down during embryonic development, they are not formed in human scar tissue when new skin

is grown. Therefore, the next important step is to identify a similar stem cell population in human hair follicles. If we can understand how these stem cell populations are regulated, then we may be able to induce proliferation to produce new hair follicles.”

Maria added: “Another application of great interest is the development of basal cell carcinoma – the most common form of skin cancer – the origins of which have not yet been determined. It is still not clear whether BCC originates in the interfolicular epidermis or the hair follicles, but using Lgr5 as a marker of hair follicle stem cells offers a potentially powerful tool to investigate the genesis of this cancer. Deregulation of the pathways that control these cells can cause sustained activation of their proliferation and disturb their normal differentiation process, which increases the risk of cancer formation.” (* Nature Genetics 40, 1291 - 1299 (2008))

The Infinite 200 and NanoQuant Plate for analizing small volumes of RNA sample

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SAMPLE MANAGEMENT TECAN JOURNAL 1/2009

Automated processing for pioneering translational medicine project Aurora Health Care is relying on its Freedom EVO® liquid handling platforms and REMP Small Size Store™ to provide automated processing, cataloging and storage of samples for an innovative translational medicine project.

Aurora Health Care is a not-for-profit health care provider, serving eastern Wisconsin in the United States. Established in 1984 by the affiliation of two Milwaukee hospitals, Aurora Health Care was founded on a simple premise; that there is a better way to provide health care. Since these early days, Aurora has expanded to serve a population of more than a million people, and now comprises 13 hospitals, over 120 outpatient clinics and approximately 140 pharmacies.

Aurora’s founding principle still remains central to its vision for health care, and has been the driving force behind establishing a translational medicine project – a fully automated repository of biological information developed over the last five years by Aurora directors Matthew and Alfred Tector. With so many facilities, in addition to community services, including visiting nurses, physicians and hospice activities, the potential influence of this venture is far reaching. The Aurora biorepository hopes to collect 50,000 samples a year, making use of surplus blood samples collected from patients for routine clinical tests that would otherwise go to waste. With such a large volume of samples for processing, automated sample handling is essential to its success.

Dr Matt Tector, explained: “The aim of the project is to allow this research to take place without changing the experience of patients within the health care system. We will be asking patients entering the system, as part of the preliminary paperwork, to consent to letting us access their medical records and use surplus blood samples. The benefit of this arrangement is that patients won’t have to give additional blood samples, so we expect a high consent rate. Potentially we could create a library on the same scale as the UK Biobank, benefiting all races and socio-economic backgrounds within our region. To process such a large number of

samples, we will need to use robotics for as much of the processing as possible. All our colleagues with experience of automated systems recommended Tecan as the right choice for liquid handling. With the Freedom EVO/REMP SSS Factory, almost the entire process will be automated.”

“The objective of establishing the Aurora biorepository is not the discovery of new drugs and therapies, but the improvement of existing treatments,“ Matt continued.

“As a health care system, our advantage is that we are not limited to investigating a single disease, like some institutions.

Our aim is to transfer information from a research environment to a clinical setting as fast as possible. We intend to have people in the front line of health care – clinicians, nurses, physicians and pharmacists – directing our research to the appropriate studies. We can then find the expertise we need to help us accomplish those goals.

If we make a discovery that is relevant to any aspect of the health care industry, our unique set-up allows us to implement those changes rapidly.”

“Our intention is to use genetic analysis to help our physicians to create personalized therapies for individual patients. We are not a clinical laboratory, however, so we will not be reporting results to directly influence an individual donor’s care. In fact, one of the tasks of the Freedom EVO will be to assign new identities to each sample so they cannot be traced back to identify the patient. We have a Freedom EVO 100 that will be responsible for checking the barcodes on samples against the database. It will then re-rack those samples from patients who have consented to this project, and assign a new identity which incorporates certain elements of their medical history. With this system we expect to be able to process several thousand samples a day. Samples will then be transferred to our Freedom EVO

SAMPLE MANAGEMENT TECAN JOURNAL 1/2009

(l to r) Matt Kirsling, information systems specialist, Alfred Tector, MD, clinical research and transplant program medical director, Natalie Polinske, research associate and Matt Tector, PhD, biorepository director

200 / REMP SSS Factory, which will isolate DNA from the samples and store them for later use. Full automation of the process should allow us to process approximately 300 DNA samples a day. We have also purchased a Safire2™ microplate reader and are hoping to incorporate some standard genetic analysis assays into the Freedom EVO 200 protocols.”

“Planning for the biorepository has been ongoing for five years, and our relationship with REMP began very close to the start

of the process. As the project has evolved, the Tecan team has always offered good insight; they have put almost as much into this project as we have. Whenever we have needed help, Tecan’s support staff has been there. As the project grows and expands to our other facilities we hope to continue our relationship with Tecan.”

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Signaling success The Center for High Throughput Cell Biology, a brand new facility at Yale University, Connecticut, is combining automation with siRNA screening techniques to investigate signal transduction pathways.

With the aim of ‘re-writing the book’ the laboratory aims to use the high throughput capacity of its Freedom EVO® workstations to conduct genome-wide screens of primarily HUVEC cells to validate new and existing data.

Professor James E Rothman, recently returning to his alma mater as Chair of the Department of Cell biology at Yale University, has been instrumental in establishing the Center for High Throughput Cell Biology, a state-of-the-art facility situated at Yale’s new research campus between West Haven and Orange, Connecticut. Professor Rothman is no stranger to high throughput screening programs, having previously been involved in the NIH Roadmap initiative as Director of the Genome Center of Columbia University in Manhattan, New York. The Center for High Throughput Cell Biology is a facility affiliated to the Department of Cell Biology, under the directorship of Dr Lars Branden – a former associate director of the Genome Center at Columbia – and is supporting its ambitious research program with provision of screening services for external laboratories.

Lars explained the aims of the Center’s research: “The basic question we are trying to answer is ‘How are signal transduction networks organized?’ The intention is to identify the signal transduction network connections between the different signaling pathways, and the components within each pathway. Our approach differs from other projects in that we are primarily looking at one specific cell type, human umbilical vein endothelial cells (HUVEC) for our internal research. This is because many of these transduction pathways have

been elucidated in universal terms, but the theoretical pathway often does not agree with experimental findings when you get down to specific cell types. We are using pooled HUVEC samples to ensure specific genetic variations do not skew our findings, and are hoping to take in all the information about signal transduction from existing databases and validate it for our experimental set-up.”

“The primary technology we are using to identify the key regulatory entities within different signaling pathways is genomewide screens with siRNAs. If we identify a particular gene we want to know more about, we can run it against all the signal transduction pathways we have established assays for. We have already developed over 150 high-content assays in-house and about 50 low-content assays. If we find a gene that perturbs a specific pathway, we then perform extensive secondary assays, ‘biological profiling’, to identify the more specific biological relevance of the gene in question. Combining these techniques to identify the key regulators of each pathway is our initial goal, however, this is really a stepping stone for trying to understand the connections between pathways. From there we can look at how this ‘transcriptional network’ varies between cell types. If you can understand these differences then this could help to explain why you get side effects from

LIQUID HANDLING TECAN JOURNAL 1/2009

a certain drug, or how paracrine signaling works in organogenesis.”

and this cannot be achieved if the process requires too much human intervention.”

Lars added: “A project of this scale requires a multi-disciplinary approach, so within the Center we have units responsible for assay development, high throughput screening and informatics. It is hard to separate the biological science from the technology for this project, as the two are intrinsically linked. The project would be impossible without automation, and Tecan really cares about what we are doing and listens to our needs. We brought one Freedom EVO platform with us from Columbia University, and this unit is dedicated to 384-well plate formats. Tecan is currently building three new platforms for us, two assay workstations and an immunohistochemistry workstation, which will all be running 1,536-well plates. These systems will greatly improve our throughput compared with our previous set-up. When first designing our system four years ago, we integrated many functions into a single platform, and we have continued down that route with our new units. The Freedom EVO platforms are very flexible, offer amazing precision and, by using a modular approach, we can optimize the space available on the deck of the workstation. The plates are all in racks to simplify transfer between workstations; the whole point of automated integration is that it gives you total control

“The new 1,536-format platforms should be able to run up to 80 plates in 24 hours and, if you consider that a genome-wide screen in quadruplicate only requires about 52 plates, this really demonstrates our potential capacity. Our new imaging workstation also has facilities to conduct both primary and secondary screening assays so, if we get a positive result, we can re-screen the well at high resolution and run secondary assays we have built into the system. This multiplex approach helps to minimize cost and increase efficiency, generating a lot of data very rapidly, and our biggest bottleneck will then most definitely be in data storage and data processing.”

“Working closely with Tecan to design this system, we have realized that we would like to take our collaboration to the next level. Tecan has believed in this work from the beginning and, even before a proposal was put to us, there was a lot of joint work done to resolve potential problems. It has to be a two-way relationship and, from the very beginning, we have been developing some new ideas together. It’s a lot easier to work with a company with whom you have established a good rapport and, with Tecan, we know that we will always get the support we need.”

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Collaboration paves the way for flexibility in diagnostic laboratories Applications specialists from Tecan Austria have teamed up with scientists from Technoclone GmbH, Vienna and the University Children’s Hospital in Zurich to set up fast and flexible microplate-based assays for a full range of benign hematology conditions in children.

The University Children’s Hospital in Zurich is Switzerland’s largest pediatric facility, and offers a spectrum of pediatric specialties ranging from emergency care through to surgery, as well as boasting an extensive research program. The hospital’s hematology division combines routine clinical diagnostics with investigation into a range of erythrocyte- and platelet-related conditions in children. Dr Oliver Speer, head of the Experimental Hematology and Erythrocyte Laboratory, explained: “Our work is focused on benign hematology.

This includes hemaglobinopathies such as thalassemia and sickle cell disease, as well as enzyme defects in the erythrocyte glycolytic pathway, such as G6PD deficiency (Favism) or ADA (important for diagnosis of certain immune deficiencies). We also look into primary and secondary hemostasis, including a major research project

examining the endogenous thrombin potential before and after therapy in paediatric idiopathic thrombocytopenia purpura (ITP) patients. We work exclusively with patient samples, performing analyses with whole blood, platelet-rich plasma, plasma or cell culture depending on the investigation.”

Many of the laboratory’s assays, for both routine diagnostics and research applications, are based on photometric techniques and historically the laboratory used different instruments for each assay.

In the last year, Dr Speer has overseen the transfer of all photometry to Tecan’s Infinite® M200 Quad 4 Monochromators™based microplate reader: “This new system is faster than our previous set-up and we can use it for all of our assays. We have gone from using 1 cm cuvettes to 96-well microplates and this gives us so much more flexibility, as well as the speed and capacity for a much higher sample throughput. The protocol for each assay is pre-set, so all we need to do is select the relevant protocol and the system automatically gives onscreen instructions. Initially we compared results between the Infinite and our older instruments and we were very soon confident enough to switch entirely to the Tecan instrument. Once we had completed transfer and validation of our existing assays, we realised that the flexibility and sensitivity of the Infinite reader would allow us to supersede some of these assays with more efficient or sensitive methods. One example of this is ferritin detection,

(front l to r) Marlis Schmid, Karin Zurbriggen, (back l to r) Oliver Speer, Alexandra Förderer, team members of the hematology laboratory

which was previously assessed using an ELISA technique. With the new system we have been able to switch from this expensive and time-consuming method to a far faster and more flexible turbidometric assay. We also became interested in new technologies such as Technoclone’s thrombin generation assay (TGA).”

Technoclone’s novel TECHNOTHROMBIN® TGA measures thrombin formation using a fluorigenic substrate and can be used to monitor hemophiliacs during inhibitor-bypassing therapy, to monitor anticoagulation therapy and to determine states of thrombophilia. Veronika Binder, CEO of Technoclone, explained: “The TECHNOTHROMBIN TGA offers exceptional sensitivity. It measures the whole kinetics of thrombin generation, not only during the initial phase of thrombin formation, but also during the phase of down-regulation and inactivation of the formed thrombin.

Dr Speer approached us requesting validation data for the assay using the Infinite system and, because so many of our customers have Tecan instrumentation, we recognized that validating it for the Infinite series readers would be of great value to many laboratories. It also offered us a rare opportunity to work with pediatric samples, so we approached Tecan and worked closely together on the validation.

Application specialists from Tecan Austria validated the instrument using control samples and soon Dr Speer was able to continue the process using patient material.

Tecan has been very good to work with, and this has been an excellent example of two companies working together for the benefit of a joint customer. Validating our assay with Tecan instrumentation really does provide customers worldwide with the best diagnostic solution.”

DETECTION TECAN JOURNAL 1/2009

Dr Speer added: “The collaboration between the companies has been perfect for us. Staff from Tecan and Technoclone came to the hospital, and we were able to set things up very easily; I have been surprised at just how quickly we have managed to complete this study. Throughout the entire process, Tecan has provided excellent support, from the initial installation of our system to further developing the software specifically for our needs. We are regularly in contact regarding technical questions and I very much enjoy working with the Tecan team.”

TECHNOTHROMBIN is a registered trademark of Technoclone GMBH.

Kinetics of thrombin generation

Leading the debate ‘Leading the debate’ is a new feature to the Tecan Journal where we invite you to share your views about trends in life sciences and the newest hot topics for our industry. Marc Feiglin, Chief Technology Officer for Life Sciences, gives us a flavor of what’s to come in forthcoming issues.

“Here at Tecan we are in an enviable position to be working closely with many leading luminaries in the life sciences world.

Most recently, in September 2008, we hosted the second annual Tecan symposium, bringing together researchers from around the world who rarely get the opportunity in their busy schedules to thrash out the issues affecting their own particular area of science. The focus of this event was biotherapeutics, stem cells, and regenerative medicine, rapidly developing areas that are grabbing the attention of life science researchers and clinicians alike. There are many outstanding questions associated with these technologies. How will the regulatory aspects for biosimilars develop compared to those that currently exist for small molecule-based therapeutics? Have we begun to see an impact yet from stem cell-based therapies? Where are the market opportunities for regenerative medicine? Will the recent political change in the US and the possibility of a major investment program there open up these areas even further? What role can companies like Tecan play in developing these opportunities further?”

“We certainly had some lively conversations on these and related topics during the three-day symposium in September and would love to hear your opinions too! If you’d like to contribute to our ‘Leading the debate’ column, send us your thoughts by email to talk@tecan.com.”

The thrombin generation cascade

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EVENTS 2009 TECAN JOURNAL 1/2009

Meet Tecan at these events in the first part of this year Americas Lab Automation 2009

Palm Springs, CA

25-27 Jan 2009

ABRF 2009

Memphis, TN

07-10 Feb 2009

MD&M West

Anaheim, CA

09- 12 Feb 2009

AAFS 61st Annual Scientific Meeting

Denver, CO

16-21 Feb 2009

Molecular Medicine Tri-Conference

San Francisco, CA

25-27 Feb 2009

60th Pittsburgh Conference on Analytical Chemistry And Applied Spectroscopy (Pittcon)

Chicago, IL

08-13 Mar 2009

AACR 100th Annual Meeting

Denver, CO

18-22 Apr 2009

34th Lorne Conference on Protein Structure and Function

Lorne, Australia

08-12 Feb 2009

Japanese Society for Bioscience, Biotechnology and Agrochemistry

Fukuoka, Japan

28-29 Mar 2009

Asia and Pacific

Europe, Middle East and Africa CHUV Research Day 2009

Lausanne. Switzerland

29 Jan 2009

MNBS Contact Meeting 2009

Røros, Norway

29 Jan 2009

LogP 2009

Zurich, Switzerland

08-11 Feb 2009

29. Spurenworkshop der Deutschen Gesellschaft für Rechtsmedizin

Munster, Germany

27 Feb 2009

Statusseminar Chip Technologies 2009

Frankfurt, Germany

5-6 Mar 2009

Laborama

Brussels, Belgium

12 Mar 2009

BioProcess

Düsseldorf, Germany

22-23 Apr 2009

AAC Spring Meeting 2009

Edinburgh, UK

27-28 Apr 2009

Tecan. For all your lab automation needs.

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8 Plus 1 Access, ABS, Gemini, Genesis RSP, GENios, HS 400 Pro, HS 4800 Pro, i-control, Instant Pipetting, MCA, MultiChannel Arm, NanoQuant Plate, OSND, PosID, Power Washer 384, Quad4 Monochromators, Safire2, Te-MO and Te-Stack are trademarks and Cavro, Freedom EVO, Freedom EVOware and Infinite are registered trademarks of Tecan Group Ltd, Männedorf, Switzerland.

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