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Thermo Fisher Scientific has 13 divisions ranging from high end instrumentation manufactured by our Scientific Instruments Division, to the reagents, tools and services from the Biosciences Division, to the vast array of manufactured and distributed products sold under the Fisher Scientific brand. In Australia and New Zealand our customers are able to access the entire range of services and products, both manufactured and distributed, from one fully integrated organisation. Our Australian and New Zealand customers are therefore uniquely positioned to fully benefit from Thermo Fisher’s position as the world leader in serving science.

Due to the technical nature of our customer’s needs it is important that we complement the breadth of our product portfolio with ready access to high quality application expertise. By segmenting our sales and customer care teams by industry, we are able to recruit and train our personnel based on the specific needs of their industry segment. In addition to these teams, you and your scientists have easy and direct access to over eighty locally based product and application specialists whose expertise is in developing the best solutions for your unique needs.

2010 was a very exciting year for us especially in the field of Healthcare, with acquisitions of Proxeon - a proteomics workflow solutions provider, Finnzymes - a supplier of integrated tools for Molecular Biology and our recent signing of an agreement to acquire Lomb Scientific, an Australian provider of chemicals and laboratory products and Dionex a leading global manufacturer of chromatography systems. We are equally excited that one of our key suppliers, Caliper Life Sciences has acquired Cambridge Research and Instrumentation, significantly expanding their product portfolio. These acquisitions further enhance our ability to provide our Life Science customers with a terrific portfolio of products.

This issue of Bio-Innovation, which focuses specifically on Molecular Biology, will give you a sense of the breadth of product and the depth of expertise which the integrated Thermo Fisher Scientific now has to offer. Enjoy!

Nan-Maree SchoerieVice President and General ManagerAustralia & New Zealand Thermo Fisher Scientific

Cover image: Colour-enhanced image of

red blood cells leaking out of a ruptured

blood vessel. This is due to a mutation in

the ephrin-B2 gene that causes the blood

vessels to be more fragile than normal leading

to an increased rate of haemorrhaging. The

fragility is due to the inadequate coverage of

the vessel by smooth muscle cells. This kind

of leaky blood vessel is frequently found in

tumours and in certain other human diseases.

Image Courtesy of : Anne Weston, LRI,

CRUK/Wellcome Images.

ContentsLife’s better in Colour – Cellular Imaging Competition

Optimised Quantitation of Marek’s Disease Virus

Optimisation of Protein Expression in an E. coli System

in vivo Optical Imaging–an Enlightening Future

Streamlining drug discovery

Recent Developments in qPCR detection chemistries

Lorne 2011

Innovations in molecular biology products & equipment

Industry Update – News Highlights from 2010

Submit with Style – MIQE Guidelines

Discovery and verification of cardiovascular & stroke biomarkers

Improving Spectral Scanning in Fluorometry & Luminometry

Accurate and Efficient Preparation of Standard Curves

Cultivation of Human Pluripotent Stem Cells

Rapid and reproducible DNA isolation

Intrawell Cell Distribution in MicroWell Edge Plates

Optimising the DNA Purification Protocol

New Western blotting kits for fast results

Direct PCR from plant tissue without DNA purification

Keep it Clean with ART® self sealing barrier tips

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Announcing… Life’s better in Colour!Thermo Fisher Scientific Cellular Imaging Competition

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Life is better in colour, and so are your cellular images. Enter the Thermo Fisher Scientific Cellular Imaging

competition and be in the draw to win your image recreated as a work of art.To be eligible, your image

needs to be created using any of our extensive range of antibodies. To enter, send your contact details

and image to [email protected], all entries must be received by the 30th of May 2011.

For full competition details, visit our website on www.thermofisher.com.au/bio-innovation

6 Bio-Innovation

Optimised Quantitation of Marek’s Disease Virus using Multiplex QPCR

IntroductionMarek’s disease, an economically-important lymphoid neoplasm of chickens, is caused by oncogenic strains of Marek’s disease herpes virus (MDV). The virus replicates in lymphocytes and the epithelia of the skin and feather tissues(1) Despite very successful vaccination with attenuated virus strains, vaccine failures do occur as field viruses evolve towards greater virulence. In the Avian Oncogenic Virus Group, led by Professor Venugopal Nair, our research is focused on mechanisms of oncogenesis, genes involved in oncogenicity and increased virulence, and mechanisms of vaccinal protection. Central to these studies is the ability to accurately quantify vaccine and virulent strains of MDV in chicken tissue samples.Traditionally, MDV has been detected by time-consuming virus isolation from blood samples. We have developed, optimised and validated a sensitive, reproducible QPCR assay for quantitation of MDV in cultured cells and in chicken tissue samples(2). By targeting serotype-specific genes for PCR amplification, we are able to distinguish between the three serotypes of MDV. MDV is a cell-associated virus and thus it is important for us to be able to quantify the virus in the context of the number of chicken cells. To achieve this, we use a duplex QPCR assay to quantify both the chosen MDV target gene (FAM-labelled probe) and the chicken ovotransferrin (Ovo) gene (Yakima Yellow-labelled probe) in a single reaction (2). By using calibrated standard curves for the virus gene reaction and the Ovo gene reaction, we are able to accurately calculate MDV genome copy number per 104 chicken cells, permitting meaningful comparison between samples(3, 4).

MethodologyApproximately 100ng sample of DNA was used for each reaction and samples were run in a 40-cycle PCR on an Applied Biosystems 7500 FAST real-time PCR system. The problem we have experienced with master mixes from other suppliers is that, when running duplex (virus gene/Ovo gene) reactions, the Ovo reaction was partially or fully inhibited in samples having high levels of virus DNA. This resulted in a falsely high value when calculating virus genome copy number per 104 cells. Dilution of the samples did not resolve the problem, and primer limitation for the virus gene reaction had a limited effect. Chicken embryo fibroblast cell culture monolayers were infected with a vaccine strain of MDV. At 0, 24, 48, 72, 96 and 120 hours post infection, the cells were harvested and DNA prepared. The samples were run in either

duplex QPCR (to detect the virus gene and the Ovo gene) or in singleplex QPCR (to detect the Ovo gene only). For two years we used the machine in ‘standard mode’ but more recently the need to increase sample throughput prompted us to start using the ‘FAST mode’. We compared Thermo Scientific ABsolute Blue master mix with a QPCR master mix from an alternative supplier (supplier Y) under standard thermal cycling conditions (Figure 1). In addition, we compared the Thermo Scientific ABsolute Fast QPCR mix with fast master mixes from two other suppliers (supplier W and supplier X) under fast thermal cycling conditions (Figure 2). Cycle threshold (Ct) values were subtracted from 40 (since a 40-cycle PCR was used) to obtain 40-Ct values (eg. Ct value = 5, 40-Ct value = 35), which are proportional to log10 amount of gene product detected.

Singleplex Assay ResultsThe performance of the master mixes in singleplex reactions was comparable, although reactions using supplier W’s fast master mix were significantly less sensitive. 40-Ct values for the virus gene (blue bars) increased with time in culture, consistent with virus replication. As expected, the level of the chicken Ovo gene, as measured by singleplex reaction (pale blue bars) remained fairly constant (Figure 1 & 2). We chose to use the ABsolute Blue and ABsolute Fast QPCR mixes for all our assays because the blue colour increased the contrast between the reagent which helped us to verify pipetting accuracy. (It is true that the blue colour aids pipetting accuracy but, for us, that is of secondary importance to the good PCR results. More than 95% of our reactions are duplex reactions. The singleplex reactions were only run as a comparison to confirm inhibition of the Ovo reaction in duplex reactions).

Duplex Assay ResultsUsing Thermo Scientific master mixes, in either standard (ABsolute Blue) or fast mode (ABsolute Fast), the level of the Ovo gene measured in duplex reaction was very similar to that measured in singleplex reaction, indicating that there was minimal inhibition of the Ovo reactions, even when high levels of virus DNA were present. Conversely, in duplex reactions with master mixes from supplier X or supplier Y, the Ovo reaction was markedly inhibited when the virus gene reaction 40-Ct exceeded a value of 19. Using master mix from supplier W, the Ovo reaction failed in all samples when run in duplex with the virus gene reaction (Figure 1 & 2).

Application

Susan J. Baigent, Avian Oncogenic Virus Group, Institute for Animal Health, Compton, UK.

7Bio-Innovation

Optimised Quantitation of Marek’s Disease Virus using Multiplex QPCR

SummaryWe found that in samples containing very high levels of MDV DNA (eg. feather tips) the Ovo reaction is often inhibited. Consequently, we prefer to use the ABsolute Blue standard master mix for these samples (Figure 3) since standard thermal cycling conditions tend to favour assays containing high levels of MDV. However, for the vast majority of our chicken tissue DNA samples, which have lower virus levels (for example spleen and blood lymphocytes), we use the ABsolute Fast QPCR master mix.

References:1. Calnek, B.W. (2001). Pathogenesis of Marek’s disease virus infection. In Hirai, K. (Ed). Current Topics in Microbiology and Immunology (pp 25-55). Springer: Berlin, Germany.2. Baigent SJ, Petherbridge LJ, Howes K, Smith LP, Currie RJ, Nair VK (2005). Absolute quantitation of Marek’s disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR. J Virol Methods 23(1):53-64.3. Baigent SJ, Smith LP, Currie RJ, Nair VK (2005). Replication kinetics of Marek’s disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation. J Gen Virol. 86(11):2989-2998.4. Baigent SJ, Smith LP, Currie RJ, Nair VK. (2007). Correlation of Marek’s disease herpes virus vaccine virus genome load in feather tips with protection, using an experimental challenge model. Avian Pathol. 36(6):467-74.

Fig. 1 (A): Thermo Scientific ABsolute Blue master mix (standard)

Fig. 1 (B): Supplier Y standard master mix

Fig. 2 (A): Thermo Scientific ABsolute Fast master mix

Fig. 2 (B): Supplier W fast master mix

Fig. 2 (C): Supplier X fast master mix

Fig. 3: Replication of oncogenic MDV in spleen and feather tips of infected chickens, measured by QPCR

Virus gene Ovo gene singleplex Ovo gene duplex

Fig. 1 (A): Thermo Scientific ABsolute Blue master mix (standard)

Fig. 1 (B): Supplier Y standard master mix

Fig. 2 (A): Thermo Scientific ABsolute Fast master mix

Fig. 2 (B): Supplier W fast master mix

Fig. 2 (C): Supplier X fast master mix

Fig. 3: Replication of oncogenic MDV in spleen and feather tips of infected chickens, measured by QPCR

Virus gene Ovo gene singleplex Ovo gene duplex

For further details, please email: [email protected]

8 Bio-Innovation8 Bio-Innovation

Optimisation of Protein Expression in an E. coli System using Thermo Scientifc MaxQ 8000 Refrigerated Stackable ShakersMark Schofeld, Research Scientist II, Thermo Fisher Scientific

Application

9Bio-Innovation

A B C Time of Flask Temp Induction Media 1 15 °C 3 Hours S rich 2 25 °C 3 Hours S rich 3 15 °C 16 Hours S rich 4 25 °C 16 Hours S rich 5 15 °C 3 Hours LB 6 25 °C 3 Hours LB 7 15 °C 16 Hours LB 8 25 °C 16 Hours LB

Half-Normal Plot

C

AC

99

95

90

80

0.00 1490.86 2981.71 4472.57 5963.42

70

50

302010

0

ABCAB

ABC

B

Half-N

ormal %

Probability

Error from replicatesA: TemperatureB: toiC: MediaPositive EffectsNegative Effects

Table 1: 3-factor 2-level experimental design

Figure 2: Half normal plot showing statisticalrelevance of the three experimental factors

Figure 2table 1

1. Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol. 1999 10(5):411-21.

For a complete copy of this article, please email: [email protected]

IntroductionOne of the most commonly used hosts for protein expression is Escherichia coli (E. coli)1, a relatively simple and well characterised system capable of producing large quantities of soluble protein in a short amount of time. E. coli does have a couple of key drawbacks: it does not support Post Translational Modifications (PTMs); and it is common for foreign genes to be poorly expressed, or for their protein products to become insoluble, forming inclusion bodies. This application note aims to optimise expression of a target protein using MaxQ® 8000 refrigerated stackable shakers to simultaneously test eight expression conditions.

ResultsThe target His-tagged recombinant protein used is a 10.5 kDa, multi-stranded ß barrel with an intervening helix insert region. This protein is commercially significant to the Thermo Scientifc Pierce product line and will be named Protein X for this study. By using the MaxQ 8000 refrigerated stackable shakers we were able to test growth temperature, length of induction, and effect of media in hours, rather than the many days normally needed to perform these studies. Table 1 shows the experimental design.

Using Design-Expert 7 Workstation, all three factors (temperature, time of induction [TOI] & media) were shown to have statistically important effects on the level of soluble Protein X expression, as judged from the half-normal plot (Figure 2).

The screening identified conditions of expression that resulted in a greater than two-fold improvement in the yield of Protein X. The super rich media had an overall lower expression yield, whereas temperature and time of induction (TOI) both had a positive effect on the soluble expression of the protein, but the effect was not cumulative. In fact, when both temperature and time of induction were increased,

the solubility of the protein dropped. When the cultures were grown in LB, both temperature and time of induction increased the yield of soluble protein. In combination, increased temperature and time of induction had an additive effect, increasing the solubility of the protein.

Discussion Troubleshooting the best expression conditions for each individual recombinant protein often requires brute force efforts to try multiple sets of variables. These solubility efforts can take weeks as variables are tried one by one.Using the shakers, cultures were grown at different temperatures and the effect of temperature, time of induction (TOI) and media on the amount of soluble Protein X produced was studied. All three factors were seen to have

statistically important effects on the level of soluble Protein X expression as judged from the half-

normal plot. Interestingly, the effect of increasing both temperature and time

of induction (TOI) was different for the two media.

This experiment not only shows the importance of optimising incubation parameters for recombinant protein expression in E. coli, but also that it can be achieved over hours rather

than days when using good experimental design, coupled

with superior equipment. Key to the success of this investigation was the

ability to design the experiment to run all flasks simultaneously using only two separate,

stackable refrigerated shakers.

The MaxQ 8000 shaker range includes both refrigerated and incubated models and can be stacked (up to 3 units high), providing excellent use of any available floor space. In addition, the slide out platform provides easy and rapid access to all the samples, during loading, unloading and induction, for example. As a result, multi-parameter, multi-level studies, such as the one demonstrated here, can be conducted very efficiently.

10 Bio-Innovation

in vivo Optical Imaging– an Enlightening Future

Non-invasive visible light imaging is now a widely accepted technology allowing researchers to follow many biological processes in healthy and diseased animal models. In this article we will specifically explore the use of optical imaging modalities throughout the drug development pipeline to highlight the unique benefits that this tool brings in terms of cost savings, process efficiencies and the insight into the mode of action. These benefits also apply to other areas of research such as oncology, infectious disease, inflammation, neurobiology, stem cell and transplantation research, cardiovascular disease and toxicology.

Optical Imaging In general, fluorescence allows the visualisation of both metabolically active and inactive cells in vivo, an analysis of compound bio-distribution and clearance, and serves as a convenient histological marker for validation studies. Fluorescence requires an excitation light to generate signal. Bioluminescence typically monitors metabolically active cells and requires interaction of the luciferase proteins with injected substrates. It can be exquisitely sensitive in the detection of labelled cells and can visualise a single cell subcutaneously. Both fluorescent and bioluminescent moieties can be genetically expressed in the cell, or injected conjugated to targeting probes in order to detect cell surface markers or protein activity in vivo. The simultaneous application of multiple reporters within a single animal allow for multiple quantitative physiological readouts and pathway delineation. In the best examples, optical reporters might signal quantitative gene expression, cell number, anatomical co-registration and 3D localisation with multiple reporters employed in the same animal.

Longitudinal Studies Studies performed non-invasively in the intact animal allow for repetitive and reproducible analysis over a time course. Repeatedly imaging the same animals in a longitudinal study allows researchers to achieve statistical significance with relatively few animal subjects and generate a more

biologically relevant understanding of therapeutic efficacy. The savings in time and animal handling are significant and the researcher avoids having to sacrifice animals at every time point. An infectious disease study that would require multiple steps of animal sacrifice, organ harvesting and bacterial plating can be imaged with 70% fewer animals. The labour (FTE) saving from harvesting and plating approaches 80% and results are visible in real time instead of waiting days for the counts of colony forming units used in a traditional assay. In addition the experimental animals are available for studies of residual disease and remission/relapse that might be extended for months.Cubist used IVIS in the pre-clinical development of Daptomycin to evaluate bactericidal activity using Staphylococcus aureus in a mouse peritonitis model. Their results are shared by Mortin et al., (Antimicrobial Agents and Chemotherapy, May 2007, p. 1787-1794). (Figure 2.)

tumour Models Monitoring tumour growth by optical imaging presents your vivarium with an even greater saving in “animal days” because of the weeks taken to monitor disease progression. With orthotopic models, the ability to monitor gene expression in the tumour reduces the biochemical workload saving up to 50% of FTE workload in harvesting and processing and 90% of immuno-histochemistry outsourcing. Optical imaging offers even greater benefits in models of disperse disease such as metastatic tumours. Sensitive bioluminescence imaging provides precise location and quantitation of metastases that would be hard to locate by traditional means and would require multiple histological samples to analyse.

Pathway Analysis An important aspect of drug development is understanding the drug’s mechanism of action. Monitoring drug effects on underlying gene regulation or cell signalling pathways in tissues presently relies on traditional methods such as dissection and histology that are often laborious and not entirely biologically relevant. Non-invasive real time analysis

Figure 1. 3D Bioluminescence

imaging of inflammation in mouse brain.

Figure 2. Daptomycin study in

mouse peritonitis model with Staphylococcus

aureus used by Cubicin.

Figure 1.

Feature Article

Figure 2.

Anna Christensen, Imaging Product Manager, Caliper Life Science

For further details, please email: Bio-Innovation@

thermofisher.com

biologically relevant understanding of therapeutic efficacy. The savings in time and animal handling are significant and the researcher avoids having to sacrifice animals at every time point. An infectious disease study that would require multiple steps of animal sacrifice, organ harvesting and bacterial plating can be imaged with 70% fewer animals. The labour (FTE) saving from harvesting and plating approaches 80% and results are visible in real time instead of waiting days for the counts of colony forming units used in a traditional assay. In addition the experimental animals are available for studies of residual disease and remission/relapse that might be extended for months.Cubist used IVIS in the pre-clinical development of Daptomycin to evaluate bactericidal activity using Staphylococcus aureus in a mouse peritonitis model. Their results are shared by Mortin et al., (Antimicrobial Agents and Chemotherapy, May 2007, p. 1787-1794). (Figure 2.)

tumour Models Monitoring tumour growth by optical imaging presents your vivarium with an even greater saving in “animal days” because of the weeks taken to monitor disease progression. With orthotopic models, the ability to monitor gene expression in the tumour reduces the biochemical workload saving up to 50% of FTE workload in harvesting and processing and 90% of immuno-histochemistry outsourcing. Optical imaging offers even greater benefits in models of disperse disease such as metastatic tumours. Sensitive bioluminescence imaging provides precise location and quantitation of metastases that would be hard to locate by traditional means and would require multiple histological samples to analyse.

Pathway Analysis An important aspect of drug development is understanding the drug’s mechanism of action. Monitoring drug effects on underlying gene regulation or cell signalling pathways in tissues presently relies on traditional methods such as dissection and histology that are often laborious and not entirely biologically relevant. Non-invasive real time analysis

of these underlying pathways using optical imaging can provide a direct readout without the need for dissection and histology. Furthermore, the use of multiple optical reporters in the same organism allows for multiple pathway readouts thereby facilitating compound efficacy analysis in a rapid and high throughput manner. Pharmaceutical companies use markers like p53, MAPK, HIF-1 or NFkB linked to luciferase as reporters in primary pharmacodynamic screens in vivo (Wang et al., PNAS, July 18, 2006, vol. 103, 11003-11008).

Molecular targeting Additional reporters that provide further context to a study could include targeted antibodies with fluorescent or bioluminescent labels. Peptides can be targeted to selectively bind to bone, metalloproteases, arterial plaque or to a site of inflammation. Spectral unmixing of the optical reporters allows removal of background autofluoresence and the resolution of five or more reporters in a single animal. With more reporters included in the study and a faster time to result there is more project time available for the medicinal chemists to optimise the compound and a higher confidence in it progressing through to the clinic.

Precise Calibration and Quantitation None of the target validation or accelerated pre-clinical testing would be of real value if the results were not absolutely quantitative. By using rigorous calibration techniques and calibrating instruments against NIST verified standards, IVIS optical data are comparable across experiments and from location to location. Importantly, they provide the dynamic range needed in a longitudinal multiple-time point drug efficacy study. Because photons are scattered in animal tissues, precise quantitation requires 3D diffuse tomography to localise and calculate the signal. This is to be contrasted with x-ray computed tomography, where the 3D representation is computed from straight- line propagation of x-rays through tissue.

In diffuse tomography, photon diffusion theory is used to model the emission of light from the probe to the animal surface and using the measured data as input, a linear system of equations is solved to determine the internal 3D distribution. In this way, a bioluminescent signal can be mapped to a tumour location in 3D and calibrated to a number of cells. Similarly, a fluorescently labelled antibody could be mapped to receptors on the same tumour and calibrated to show the number of dye molecules. The 3D representation can be co-registered to an anatomical atlas, or to imaging data from a different modality such as MRI or CT.

Summary Some of the most exciting drugs approaching the clinic today – in areas as diverse as oncology, infectious disease or osteoporosis – are getting there sooner because of optical imaging techniques using IVIS from Caliper Life Sciences. Cubist used IVIS in the development of Daptomycin, Pfizer published on IVIS in the development of Sutent, Novartis used IVIS with CHIR51 and Amgen used IVIS to investigate RANKL inhibitors during the development of Denosumab. In fact, optical imaging using IVIS technology contributed to the development of more than 14 drugs currently in development or in the clinic and IVIS users report three major benefits:

Efficiency – Optical imaging has high enough throughput to support animal studies early in drug development. Results can take half the time of traditional approaches providing rapid progress toward clinical trials with a more robust drug candidate.

Economy – Longitudinal studies using optical reporters provide more rigorous data from fewer animals than traditional methods. A study with up to 70% fewer animals saves on compound synthesis, has less FTE time, fewer analytical steps and lower outsourced histology costs. Longitudinal studies provide robust statistics and results can often be seen earlier than with traditional histology.

Insight – Optical imaging can provide you with mechanistic insight into the mode of action, or with dosing and treatment models to inform your clinical trials. Co-registering optical data with traditional imaging modalities like CT provides the final translational step to the clinic. As a leader in biology, physics and instrumentation, Caliper Life Sciences now provides the know-how, equipment and biological tools for non-invasive visible light imaging. Caliper offers a suite of imaging tools to meet your needs from luciferase-labelled bacterial or cell lines, through to imaging systems or support services to suite your optical discovery program.

Figure 3. Inflammation in arthritis model using bioluminescence and X-rayon IVIS.

Non-invasive visible light imaging is now a widely accepted

technology allowing researchers to follow many biological

processes in healthy and diseased animal models.

11Bio-Innovation

Figure 3.

12 Bio-Innovation

Streamlining drug discovery– Reducing false results with accurate dispensing

High throughput screening (HTS) has enabled the successful implementation of large-scale biochemical assays. As a result, drug discovery researchers have been able to rapidly identify active compounds, antibodies or genes of interest for downstream development. These high throughput assays are commonly incorporated upstream in the drug discovery process in order to ensure that potential candidates, with minimal off-target effects are accurately identified, thus saving valuable time and resource. In addition, they are often incorporated as the starting point for drug design, to provide a more in-depth understanding of both intra-cellular and inter-cellular interactions.

Introduction HTS provides an accurate and reliable method of identifying and analysing cellular events. By screening a wide range of static assays against a comprehensive library, researchers can effectively identify and analyse cellular events, such as kinase activation/inhibition, up/down regulation of signal transduction pathways and apoptosis. There are a vast number of specific assay types, and in order to perform a precise screening campaign, the assays used depends on the therapeutic targets. However, the reliability of any assay is dependent upon a precise method of liquid dispense. In order to reduce the chances of incurring any error, it is vital that liquid volumes are exact.The need for such an accurate dispensing of liquid volumes has ensured that multichannel liquid dispensers have become commonplace within the majority of laboratories. Such methods of liquid transfer are often used in the dilution of concentrated stock solutions, for subsequent dispense across the assay plate, where they will be analysed. Yet there is the potential for incurring errors when creating any dilutions, or when dispensing compound solutions into the assay plate. These inaccuracies will consequently propagate into false positive or negative results during the initial screen.

With additional uses in the pharmacological proofing of compounds, liquid handling instrumentation has the capability to accurately perform repeated dispensing, mixing and aspiration cycles in successive wells. The resulting drug titrations or dose response curves can thus be used to determine drug candidate potency. Accuracy is therefore instrumental in the creation of these dilution series to ensure that the most potent compounds are selected and moved forward for further analysis. The time required for hit-to-lead and lead-to-market is thus minimised, saving laboratory resource.

Minimising errors with minute volumes Due to the small, often microliter, liquid volumes typically handled during HTS, inaccuracies of just 1µL can have a significant impact on experimental integrity. It can cause the production of false positive or negative results. Although this is not necessarily detrimental to the resulting data itself, false positives will often reduce laboratory efficiency and cause a degree of frustration. By minimising the number of false positives, the number of time-consuming re-tests and reagent loss will be reduced. False negatives can also lead to lost opportunities, where potential lead compounds have been dismissed due to inaccurate data. As such, this can lead to a requirement for more regular and extensive re-testing to identify any potential lead compounds, which have been missed. In order to maximise efficiency and ensure cost-effectiveness, researchers need to be sure that all dispensed volumes are highly accurate. In order to demonstrate this, we investigate the dispensing precision of microliter volumes using the Thermo Scientific Versette liquid dispensing automation platform. Its performance was validated across a wide volume range using a single, 8, 12 and 96 channel pipetting head to be certain of accuracy and precision over the instrument’s complete range of capabilities.

Application

13Bio-Innovation

System verification Liquid transfers were evaluated using ten previously determined (1) optimisation parameters: air gap; blow out; aspiration and dispense speeds; dwell times; neat/incremental dispense; overstroke; tip height; tip touch; offset move command; and volume correction. Each of these parameters was subsequently tested using a multichannel verification technique – a dual dye system which has been widely used in the automation field to calibrate liquid volumes from a variety of automation platforms. This system offers a methodical validation of the liquid handling instrument and includes: dye solutions (sample and diluents), a calibration plate, an orbital plate mixer, as well as spectrophotometric read-out and data analysis software. Three different volumes (2, 3 and 30µL) were dispensed using the single, 8, and 12 channel heads to evaluate accuracy over a wide range. The 96 channel head was then used to dispense five volumes, from 0.5 to 30µL, to determine that this versatile liquid handler can dispense a broad spectrum of liquid volumes in a highly precise and accurate manner. The assessment of these ten parameters is critical for the generation of reliable data when incorporated into automated workflows.

Accuracy was confirmed via the following process: • Dyewasdispensedattheselectedvolume

into a 96-well plate • Theplatewasshakenat1000rpmforthreeminutes

and centrifuged at 1700rpm for one minute • Spectrophotometricabsorbancewasmeasuredat412nm

Following this, the coefficient of variation (%CV) and margins of error (%error) were calculated using the multichannel verification system software.

Results The data obtained (2), including the %CV and %error, were directly compared for a wide range of volumes using the 1, 8, 12 and 96-channel pipetting heads, as shown in table 1. This data demonstrates that comparable results were obtained using the Versette and the multichannel verification system. The volumes dispensed from this versatile liquid handling platform are highly accurate and precise, making it a reliable system for the dispense of minute volumes. This is, in part, due to the advanced pipetting mechanics, which are easily adjustable for improved accuracy. With five pre-calibrated liquid classes for fast adjustment between standard solutions, performance can easily be optimised when switching between liquid types. As a result, experimental error experienced with screening assays due to dispensing inaccuracies is significantly reduced.

Conclusion As a vital part of any HTS protocol, automated liquid handling is integral to the drug screening and thus drug discovery process. In order to maintain experimental integrity and maximise laboratory efficiency, researchers need to ensure that precise data is obtained from every screen. Thus, all potential sources of error must be significantly reduced, if not completely eliminated. Ensuring the accuracy and precision of all dispensed liquid volumes is therefore of extreme importance. As demonstrated here, the modular Thermo Scientific Versette automation platform for liquid handling provides the functionality to dispense volumes from 0.1 to 1250µL in an extremely precise and accurate manner. Compatible with nineteen interchangeable pipetting heads, from single to 384 channels, a wide variety of throughput requirements are easily met. As such, when used in the creation of a dilution series, users can be confident that the occurrence of false positive or false negative results is significantly reduced, while throughput is maximised without any compromise in precision.

1. Optimising low volume transfer using automated liquid dispensers.

Thermo Fisher Scientific application note ALH_09002.

2. Tal Murthy, Brian Hewson, Kiara Biagioni and Zoltan Brutler.

Modularity of Thermo Scientific Versette automation platform

and its performance with ARTEL MVS system.

Poster presented at SBS March 2010.

High throughput screening (HTS) has enabled the successful implementation

of large-scale biochemical assays. As a result, drug discovery researchers

have been able to rapidly identify active compounds, antibodies or genes

of interest for downstream development

Table 1: The preliminary performance data obtained from the Thermo Scientific Versette. Performed at the Applications laboratories of Thermo Fisher Scientific

Volume (µL) %CV %Error

1 channel

2 5.3 5

3 1.1 3.8

30 0.7 1.5

8-channel

2 6.4 9.6

3 5 1.1

30 1.3 1.9

12-channel

2 2.6 5.3

3 1.8 4.1

30 0.6 1.2

96-channel

0.5 12.6 12.2

3 1.4 3.6

7 0.5 1.2

14 0.6 0.5

24 0.5 0.9

30 0.8 1


14 Bio-Innovation

DNA Detective – Recent Developments in qPCR detection chemistries

IntroductionIn real-time qPCR, the target DNA sequence is amplified and simultaneously quantified throughout the amplification reaction, during each PCR cycle. In a perfectly designed assay, when the amplification reaction is in the log (linear) phase, the quantity of the PCR product is directly proportional to the amount of input nucleic acid. Accumulation of the target DNA sequence in real-time qPCR is detected and measured using a fluorescent reporter molecule. As the quantity of target amplicon increases, so does the intensity of fluorescence emitted from either a DNA-intercalating dye or from a probe that is specific to a sequence within the amplicon. Reactions that contain a higher concentration of the target sequence take fewer cycles to accumulate a threshold concentration of PCR product, while those containing less template require more cycles; the threshold being the point at which the fluorescence resulting from amplified product is detectable above background fluorescence. The number of PCR cycles that elapse before the threshold is reached (Cq) is a measure of the input nucleic acid (Figure 1). By comparing the results of samples of unknown concentration with a series of standards, the amount of template DNA in an ‘unknown’ reaction can be accurately determined, this approach being referred to as absolute quantification. The quantity of target nucleic acid can also be measured using relative quantification which measures the ratio between the target nucleic acid and one or more reference genes. As the method has developed, qPCR has become one of the most powerful and sensitive gene analysis techniques available. It is used routinely for the quantification of both DNA and RNA (following reverse transcription to cDNA) in a broad range of applications in industrial, academic and diagnostic laboratories and has become a routine method for measuring the expression of genes of interest, validating microarray experiments, monitoring biomarkers and measuring genetic variations (SNPs).

qPCR detection chemistries There are two main types of fluorescence-based detection chemistries that are commonly used in qPCR: intercalating dyes and sequence-specific DNA probes. Changes in the fluorescent signal, resulting from the accumulation of target amplicon, are measured using a fluorescence detection system in the qPCR instrument.

Intercalating dyes Intercalating dyes, such as SYBR Green I, are non-specific fluorescent dyes that intercalate with double stranded (ds) DNA during PCR. Following primer-mediated replication of the target sequence, the dye molecules bind to the dsDNA product and emit a greater fluorescent signal during excitation compared to the free dye in solution. As the quantity of target DNA (or cDNA) increases during PCR, so the intensity of fluorescence increases. There are various reasons why researchers may wish to focus on the use of intercalating dyes but there are also issues associated with their indiscriminate binding to dsDNA and quantification of non-specific amplification artefacts (such as primer-dimers) contributing to inaccurate quantification of target sequences. Intercalating dye technology is not the focus of this article.

Sequence-specific DNA probes Sequence-specific DNA probes are oligonucleotides that are usually labelled with a fluorescent reporter molecule at one end and a quencher molecule at the other. The probe contains a sequence that is complementary to the target DNA sequence. Since their target sequence is designed to be within an amplicon, this increases the specificity of the fluorescent signal and the accuracy of the quantification, even in the presence of non-specific DNA amplification. There are several different types of fluorescent reporter probes. Some of the more complex probes contain a hairpin loop structure, such as Scorpion™ probes, where a complementary sequence (a PCR primer) is attached to one end of the stem, or molecular beacons, where the complementary sequence is contained within the loop. When a probe containing a hairpin loop is free in solution, the stem of the structure brings the fluorophore in close proximity to the quencher, resulting in quenching of the fluorescent signal. However, when the complementary sequence binds to the target DNA, the hairpin loop structure is disrupted and the fluorophore is separated from the quencher, resulting in a corresponding increase in fluorescence. Alternatively, probes can have a linear structure, such as TaqMan (Applied Biosystems) or a randomly coiled structure, such as Thermo Scientific Solaris. The fluorescent signal results from hydrolsis of the probe, following hybridisation, or as a result of hybridisation to the target sequence.

REFERENCES 1. Saiki, RK, Scharf S, Faloona F, Mullis KB,

Horn GT, Erlich HA and Arnheim N. (1985)

Enzymatic amplifcation of beta-globin genomic

sequences and restriction site analysis

for diagnosis of sickle cell anemia. Science

230(4732): 1350–1354.

2. Lukhtanov EA, Lokhov SG, Gorn VV,

Podyminogin MA and Mahoney W. (2007)

Novel DNA probes with low background and high

hybridisationtriggered fuorescence. Nucleic

Acid Research 35(5) e30.

3. Lukhtanov EA, Lokhov SG, Gorn VV,

Podyminogin MA and Mahoney W. (2007)

Novel DNA probes with low background and high

hybridisationtriggered fluorescence. Nucleic

Acid Research 35(5) e30.

qPCR is a technique that gathered momentum during the 1990s and has now become a standard method used by most molecular biology laboratories. Some more recent developments in probe technology are discussed in relation to Thermo Scientific Solaris.

Application Tobias Hampshire Ph.d | European Pcr Product Marketing

Manager Thermo Fisher Scientific

15Bio-Innovation 15Bio-Innovation

DNA Detective – Recent Developments in qPCR detection chemistries

Figure 1 : Ten-fold dilutions of cDNA synthesised from synRNA amplicon sequence was amplified on an ABI 7900HT Instrument using the Solaris qPCR Assay of CDC20. The log-scale amplification curves are shown along with the performance of the assay including efficiency, r2 value, dynamic range out of 10 log 10 dilutions and the lower limit of detection.

Fluorescence on hydrolysis (taqMan) The fluorescent reporter and the quencher are maintained in close proximity while the probe is intact. The probe is designed to anneal to the target sequence between the forward and reverse primer sites. It is then hydrolysed by the 5’-3’ exonuclease activity of Taq DNA polymerase, disrupting the proximity of the fluorophore and the quencher, and resulting in an increase in fluorescence. If no target PCR product is present, the probe is not degraded and the fluorescent reporter remains quenched.

Fluorescence on hybridisation (Solaris) When the probe is free in solution the randomly coiled structure brings the fluorescent reporter and the quencher together. Hybridisation of the complementary sequence to target DNA during the annealing step increases the distance between the dyes, resulting in an increase in fluorescence released intact into the solution, where the fluorescent signal is once again quenched, allowing it to be used in subsequent cycles. Continued on page 16.

table 1. Solaris design algorithm parameters 1. When designing a functional assay, numerous design parameters are applied with high stringency including: overall GC content, optimal sequence length, melting temperature, stretches of homogenous nucleotides (eg GGGG). 2. The algorithm adjusts the Tm and enables universal cycling conditions by incorporating the MGB moiety and by selective placement of Superbases. 3. When there is more than one splice variant for a target gene, a consensus (or common) sequence is identified, representing a design space that produces an assay which can detect all known splice variants. 4. BLAST analysis is a critical component of any comprehensive qPCR assay design protocol. The algorithm utilises genomic, transcript and pseudogene databases to identify and eliminate sequences that are more likely to lead to erroneous priming and detection (ie. off-target effects). 5. To mitigate the potential for genomic DNA amplification, the design algorithm, whenever possible, will place one of the assay components (probe or primer) or amplicon over an exon junction boundary.

Figure 1 :

16 Bio-Innovation

Minor Groove Binder technology A recent development in the design of fluorescent reporter probes is the utilisation of minor groove binder (MGB) technology. Examples of fluorogenic MGB probes include MGB-TaqMan (Applied Biosystems), MGB-Eclipse® (Sigma-Aldrich) and Solaris (Thermo Fisher Scientific). MGBs are flat, crescent-shaped molecules that have a natural ability to fold back and fit snugly into the minor groove (the deep, narrow groove between the two phosphate-sugar backbones) of the dsDNA helix. This provides an extremely stable hybridisation between the DNA probe and the target sequence. The stability of this hybridisation increases the temperature needed to melt or dissociate the probe from its target (Tm), allowing shorter probe sequences to be used. Not only does this improve mismatch discrimination, but it also permits more efficient probe design.(3) Prior to MGB technology, the size of designed probes had to be larger in order to produce melting temperatures consistent with efficient PCR. Such long probes reduce design flexibility, when restricted by small target regions, and are less sensitive to mismatch discrimination.

Superbase technology The strength of Superbase technology has been harnessed in the development of Solaris Probe Assays (Thermo Fisher Scientific). Often, the sequence of the target DNA region can affect the sensitivity and specificity of primer probe designs. For example, target sequences that are rich in A-T pairs often have lower melting temperatures and G-rich regions can be affected by guanine-guanine self association. Superbases are modified derivatives of native nucleotides that, when substituted in primer and probe designs, can eliminate many of these problems and maximise the design space within a particular sequence. Super A and Super T improve the stability of traditionally weaker A-T bonds,

enabling the Tm to be raised for more efficient hybridisation and improving the performance of assays in A-T rich regions. Super G eliminates guanine-guanine self-association that interferes with proper hybridisation in G-rich sequences. The use of Superbases can increase the flexibility of probe/primer design algorithms, allowing otherwise difficult probe and primer sequences to become viable genomic assays. They allow the algorithm to refine the Tm, reduce secondary structures and improve mismatch discrimination. The use of both MGB and Superbase technology in fluorogenic probes serves to adjust and standardise melting temperatures, allowing assays to be performed optimally under the same thermal cycling conditions. This is often referred to as ‘universal thermal cycling conditions’ and reduces the necessity for preliminary optimisation of assay conditions.

Design algorithms A robust primer/probe design algorithm is necessary to ensure optimal functionality, specificity and splice variant coverage (Figure 2). This is particularly important in gene expression assays where, previously, a significant amount of time was spent in selecting and optimising the primer/probe set. By incorporating important design rules (Table 1), it is possible to design a single high performance assay for specific gene expression experiments.

Gold standard method Advancement and utilisation of the technologies above have enabled qPCR to become the gold standard method for the validation of a wide-spanning number of scientific and medical approaches from the detection of pathogens in clinical specimens, the monitoring of genetic disease progression and therapeutic effect to the validation of data generated from microarray and RNAi based experiments in more fundamental research projects.

“The use of Superbases can increase the flexibility of probe/primer design algorithms, allowing otherwise difficult probe and primer sequences to become viable genomic assays”

EXON 1 EXON 3 EXON 4 EXON 5 EXON 6 EXON 7

Genomic DNA

Splice Variants

EXON 3EXON 2EXON 1 EXON 4 EXON 6 EXON 71

PRIMERPRIMER

EXON 3EXON 2EXON 1 EXON 5 EXON 62

PRIMERPRIMER

EXON 2EXON 1 EXON 5 EXON 63

PRIMERPRIMER

EXON 2

PRIMER

PRIMER

Forward Primer

Probe

Reverse Primer

EXON 1

Genomic DNA

Splice Variants

1

Figure 2: Solaris qPCR Assays are designed

to a consensus sequence among all

known splice variants so one assay provides

comprehensive results.

Application


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Industry Update News Highlights from 2010

Dec 13, 2010. Thermo Fisher Scientific to Acquire Dionex Corporation. Dionex, based in Sunnyvale, Calif., is a market leader in liquid chromatography and extraction systems with more than 1,600 employees in 21 countries spanning six continents, including a significant presence in the Asia-Pacific region. “We believe the combination of Thermo Fisher and Dionex is extremely compelling from a technology, market and financial perspective,” said Marc N. Casper, president and chief executive officer of Thermo Fisher. “Dionex’s strength in chromatography instruments, software and consumables complements our leading positions in mass spectrometry and laboratory information management systems.

Apr 15, 2010. Thermo Fisher Scientific Inc. (NYSE: TMO), announced today that it has acquired Proxeon A/S, an innovative supplier of products for proteomics analysis headquartered in Odense, Denmark. The company is recognized for its ability to provide a simplified proteomics workflow, including nanoflow liquid chromatography systems, columns, ion sources, and bioinformatics software, to meet the need for robust high-sensitivity liquid chromatography/mass spectrometry (LC/MS) analysis in complex proteomics applications. Proxeon’s nanoflow liquid chromatography system, EASY-nLC, has been adopted by a number of the world’s leading proteomic centers for its exceptional simplicity and ease of use.

Feb 01, 2010. Thermo Fisher Scientific Inc. (NYSE: TMO), announced today that it has signed a definitive agreement to acquire Finnzymes, a well-recognized provider of integrated tools for molecular biology analysis, including reagents, instruments, consumables and kits. Finnzymes provides comprehensive solutions for high-performance polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and real-time quantitative PCR (qPCR). The company’s expertise in DNA polymerases has led to significant increases in the performance of these enzymes, with the ability to quickly and reproducibly amplify and quantify particular DNA sequences which are routinely used in many molecular biology applications.

Dec 21, 2010 -- Caliper Life Sciences, Inc. (NASDAQ: CALP), a leading provider of products and services for drug discovery research, announces that it has completed the acquisition of privately-held Cambridge Research & Instrumentation, Inc.”We are excited to have completed this transaction which adds CRi’s proprietary multiplexed in vivo and tissue imaging technology to Caliper’s leading portfolio of drug discovery, imaging and diagnostics solutions,” said Kevin Hrusovsky, President and CEO of Caliper Life Sciences. “CRi’s products provide an entry point for Caliper to address the expanding billion-dollar tissue imaging and digital pathology clinical research market.”

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Nov 30, 2010. Thermo Fisher Scientific Inc. (NYSE: TMO), announced today that it has signed a definitive agreement to acquire Lomb Scientific, a well-known provider of laboratory chemicals, consumables and instruments to leading hospitals, universities, research and analytical laboratories in both Australia and New Zealand. “The addition of Lomb Scientific reinforces our commitment to expand in growing Asia Pacific markets,” said Marc N. Casper, president and chief executive officer of Thermo Fisher Scientific. “Through this acquisition, we would significantly strengthen our laboratory product offerings in the region, particularly chemicals used in life sciences, research and industrial applications.”

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Bio-Innovation 25

In recent years there has been a lack of consensus within the scientific community with respect to experimental design, data reporting and analysis of qPCR experiments. Unfortunately, the absence of several experimental standards has raised concerns within the scientific research community over the reliability of qPCR data interpretation. In an effort to provide such standardisation when reporting qPCR results, key opinion leaders in the qPCR community recently published a set of guidelines known as “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments”. The aim of this publication is to provide a benchmark for the quality assessment of a qPCR assay reported in a given publication.

the Essential SelectionThe MIQE guidelines now define the minimum information required for evaluation of qPCR results, and include a checklist to be included in the initial submission of a manuscript to a publisher. That checklist includes 85 specifications of which 57 are deemed essential, in that they must be reported on to ensure the relevance, accuracy, correct interpretation, and repeatability of a qPCR experiment. Briefly, the central tenets within the MIQE publication involve adoption of standardised nomenclature (e.g. the use of the quantification cycle (Cq), rather than threshold cycle (Ct), crossing point (Cp), or take-off point (TOP)) and considerations of several other issues including:

• Reportingontheanalyticalsensitivityorminimumnumber of copies that can be accurately measured for a given assay;

• Theanalyticalspecificity,wherebyonlytheappropriate target is amplified and avoiding other non-specific targets or gDNA that may be present;

• Theoverallaccuracyreferringtothedifferencebetweenexperimentally measured and actual concentrations;

• Intra-assayrepeatabilityand• Inter-assayreproducibilityreflectingtheprecision

and robustness of the assay.

Now, both experienced and new users to the qPCR arena can benefit from adopting these best practices to enhance the quality and consistency of data reported in the literature, and longer term, it is likely that journals will make MIQE a basic submission requirement. Thermo Scientific Solaris qPCR Gene Expression Assays are provided with sequence information, and are highly repeatable, and specific to provide confidence in submitting results for publication. For more details please email [email protected]

Submit with Style

The MIQE Guidelines were first published early in 2009 and are a recommended set of guidelines, or minimal information requirement, for the submission of scientific papers containing qPCR data.

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Bio-Innovation26

Groundbreaking research leading to the discovery and verification of cardiovascular & stroke biomarkers

Professor MingMing Ning Massachusetts

General Hospital Harvard Medical

School Professor of Neurology Director Clinical Proteomic Research Center

Thrombolysis with intravenous (IV) tissue plasminogen activator (tPA) is currently the only FDA-approved medical therapy for acute ischemic stroke. Although efficacious in increasing the proportion of patients with better neurologic outcome by 30-35% at 12 months, tPA is only given to less than 5% of stroke patients, due to its dreaded side effect of 10-fold increased risk of intracranial hemorrhage (ICH), with more than 50% mortality rate for patients with major hemorrhage.1

tPA, a serine peptidase, has pleiotropic effects regulating the expression of matrix metalloproteinases (MMPs) and other proteases involved in cell-cell signaling in the brain. For example, tPA may increase the expression of matrix metalloproteinase 9 (MMP-9), which is implicated in the breakdown of the blood-brain barrier leading to ICH.2

While previous studies of bedside biomarkers have been limited to known individual factors, such as MMP-9, the MMP-tPA axis has a variety of pleiotrophic interactions.3

New proteomic methods and technology may help to identify both known and unknown factors at the same time, perhaps improving our understanding of thrombolytic therapy with respect to protease and cell-signaling interaction, a concept particularly enticing for disease entities such as stroke, which most likely involve multiple gene interactions.4

One novel method in particular is the study of protease “degradomics” or substrate profiling: the investigation of, and techniques for characterising, the “substrate repertoire” of proteases of interest.5, 6 This method is particularly attractive to the study of multiple protease interactions and allows us to understand the biologic role of proteases both from an individual and global perspective.

A research team led by Professor Mingming Ning, Director Clinical Proteomic Research Center at Massachusetts General Hospital (MGH), recently published their findings describing their first attempt of proteomic substrate profiling (degradomics) in stroke patient plasma, (Figure 1) to understand better the effect of tPA on cell-cell signaling in the brain in the context of acute stroke pathophysiology, in order ultimately to elucidate the mechanisms of the therapeutic efficacy and side effects of thrombolysis7.

Significant advancements in the use of tissue plasminogen activator (tPA) in stroke treatment have also been facilitated through the collaboration between the Thermo Fisher Scientific Biomarker Research Inititatives in Mass

Spectrometry Center (BRIMS) and Harvard University. tPA is a drug that can be administered within a three-hour window from when stroke symptoms occur. The treatment, which works by dissolving blood clots, has proven highly efficacious but involves significant risks. Only 5% of patients fit the timeframe criteria within which it is safe to administer tPA. Through the use of mass spectrometry-based proteomics workflows, (Figure 2) data from the collaborative research has helped scientists identify a wider scope of patients who may benefit from tPA regardless of the timeframe for administration. As part of the research, MGH created a space to house Thermo Scientific mass spectrometers near patient bedsides. This opens the door to the possibility of obtaining and processing samples in as little as 10 minutes, enabling appropriate treatment to be rapidly administered.

Dr. Ning comments: ‘We are now able to conduct research in real time, directly at the beside of acute stroke patients, thanks to the collaboration between Dr. Mary Lopez and her team at the BRIMS Center, Dr. Tom Jacobs at NIH/NINDS, and Dr. Eng H Lo, Dr. Ferdinando Buonanno and Dr Anne Young at the Clinical Proteomics Research Center at MGH. This has not been possible before and has crucially allowed us to build a bridge from research to treatment, enabling us to gather key information exactly when we need it to aid our stroke patients.”

1.Tissue plasminogen activator for acute ischemic stroke. The national institute of neurological disorders and stroke rt-pa stroke study group. N Engl J Med. 1995;333:1581-15872.Castellanos M, Leira R, Serena J, Pumar JM, Lizasoain I, Castillo J, Davalos A. Plasma metalloproteinase-9 concentration predicts hemorrhagic transformation in acute ischemic stroke. Stroke. 2003;34:40-463.Wang X, Lee SR, Arai K, Tsuji K, Rebeck GW, Lo EH. Lipoprotein receptormediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003;9:1313-13174.Heiss WD. Experimental evidence of ischemic thresholds and functional recovery. Stroke. 1992;23:1668-16725.Lopez-Otin C, Overall CM. Protease degradomics: A new challenge for proteomics. Nat Rev Mol Cell Biol. 2002;3:509-5196.Liotta LA, Ferrari M, Petricoin E. Clinical proteomics: Written in blood. Nature.2003;425:9057.MingMing N, Sarracino DA, Buonanno FS, Krastins B, Chou S, McMullin D, Wang X, Lopez M, Lo EH. Proteomic Protease Substrate Profiling of tPA Treatment in Acute Ischemic Stroke Patients: A Step Toward Individualising Thrombolytic Therapy at the Bedside. Transl. Stroke Res. 2010; 1:268–275

Professor MingMing Ning Massachusetts General Hospital Harvard Medical School Professor of Neurology Director Clinical Proteomic Research Center

Feature Article

Bio-Innovation 27

Figure. 2The Biomarkers Research Initiatives in Mass Spectrometry (BRIMS) Center is a Thermo Fisher Scientific center of excellence that develops and promotes comprehensive, integrated, and robust mass spectrometry (MS)-based workflows that link early-stage discovery to next-stage quantitative verification of protein, peptide and small molecule biomarkers.

Figure. 1 UV 214 absorbance chromatogram (degradomic region shown in brackets). (A) Control plasma without strokes had a stable substrate profile over time. (B) In acute stroke patients, there are differential expression patterns of smaller proteins as the stroke progresses over time. (C) tPA-treated stroke patients demonstrated different substrate patterns over time from those of untreated

28 Bio-Innovations

Improve the Sensitivity, Dynamic Range and the Accuracy of the Spectral Scanning in Fluorometry and Luminometry

When fluorescence signals are measured the results are normally referred as “Relative Fluorescence Units” (RFUs) to emphasis the fact that resulting numeric values are heavily dependent on the settings of the instrument electronics, mainly PMT gain voltage. Results are also somewhat dependent on the environmental aspects, like ambient temperature that affects the electronic noise levels. The practical effect of this relative nature of fluorescence measurements is that result values become quite difficult to compare, especially if the results are from different measurements done with different instrument settings.

Most of the available instruments partially overcome these difficulties by preventing the use of different electronic settings inside one assay, for example PMT gain voltage is fixed within an assay. This method causes new difficulties that are seen for example as a narrow dynamic measurement range caused by fixed gain voltage. When high intensity Xenon flash lamps are used for the excitation, the available dynamic range with one gain voltage is commonly reduced to about 3 – 4 orders of magnitude. Fluorometric assay chemistries offer chemical concentration ranges around 5 – 6 orders of magnitude, or even more, so the dynamic range of fixed gain voltage is not sufficient.

If a Xenon flash lamp is used for the excitation it is necessary to be able to use several PMT gain voltages within one assay to get good assay performance; both high sensitivity and large dynamic range. When an instrument with fixed PMT gain is used to measure an assay where the concentration range exceeds the available dynamic range, either high concentration samples are saturated or low concentration samples are not separated from the background, depending on the PMT gain value selected. In both cases certain samples will give incorrect result values. Thermo Scientific Varioskan Flash® has an in-built calibration system for all measurement technologies covering all mentioned problems in measurements. This paper shows how detection systems for fluorescence intensity, time resolved fluorescence and luminescence are calibrated to ensure the best possible performance in each individual measurement. Optical DesignThe basic optical design of the Varioskan Flash reader is shown in Figure 1. The reader has two optical modules. LumiSens module is used for normal luminescence measurements without any wavelength selection and for luminescence assays where filters are used to separate two or more wavelengths, the second is a monochromator based spectral scanning module that is used for all the other detection technologies. Both LumiSens module and spectral

scanning module have similar calibration systems that make it possible to use several different PMT gain voltages to measure one set of samples.

The spectral scanning module includes two reference chips in the plate carrier that produce the calibration signal, one for top reading optics and another for the bottom reading optics. These chips produce stable emission signal over the whole spectral range and are used to calibrate four predefined PMT gain voltages. Luminometric measurement module is calibrated using stabilised 560 nm green LED that can produce stable light signal over large intensity range.

Calibration processThe calibration process is divided into three phases: Initial factory calibration, start-up calibration and runtime calibration. Factory calibration process is the main system for the instrument calibration and it is fully integrated process that is completely based on these reference samples inside the unit. During this process, the instrument will measure all possible information about the instrument electronics, mechanics and optics, for example PMT sensitivity curves, lamp intensity and spectral characteristics, monochromator relative sensitivity spectra etc. This factory calibration takes about an hour. All this information is stored inside the instrument memory. Factory calibration needs to be done only after the instrument has been assembled in the factory, or if the internal software is changed or major service has been done.Start-up calibration is performed every time

Spectral scanning module

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Figure1. Basic design of Varioskan Flash spectral scanning reader

Figure 1.

Application

Reija-Riitta Harinen and Jorma Lampinen, Thermo Fisher Scientific, Sample Preparation and Analysis, Vantaa, Finland

29Bio-Innovations

the instrument is switched on to eliminate possible effect of environmental effects or aging of the electronics. This process is a shortened version from factory calibration, where the instrument checks that all calibration values are still the same as they were during the factory calibration.

Runtime calibration is performed before and during each measurement run. It includes the calibration of PMT gain voltages for those wavelengths that will be used in the measurement with spectral scanning module. The calibration has the following steps:

Before the measurement: PMT gain voltage calibration with the reference signals. The reference chip (when spectral scanning module will be used in assay) or the 560 nm LED (when LumiSens module will be used) sends calibration signals with certain intensities covering the whole dynamic range. All those wavelengths that have been selected to be used in the assay are calibrated using the reference signals.

These reference signals are measured with predefined (during factory calibration) PMT gain voltages. Based on this calibration data, the instrument will form a conversion table where relative differences between the PMT gain voltages are normalised and then defines light intensity levels where each PMT gain voltage should be used. This conversion table is then used to normalise measurement results that have been measured with different gain voltages.

During the measurementXenon lamp intensity calibration: When Xenon lamp flashes, each flash is an individual phenomenon, every flash has a little different intensity, flash time etc. Therefore, a small portion of the flash is directed directly to the reference detector that will analyse flash properties (intensity, length etc.). This information is used to eliminate the excitation intensity variations.

PMT gain voltage selection for each sample: The optimal gain voltage is selected individually for each sample well using one flash test measurement. Based on the emission signal level from this trial flash measurement, the Varioskan Flash selects the optimal gain voltage and performs the actual measurement. When next sample is measured, similar trial flash measurements are performed and a new gain is selected for the new sample. This trial flash test measurement is performed before each measurement in all measurement types, end-point, kinetic and spectral scanning.

Spectral CorrectionEfficiency of any detection system is affected by the electronic components included, especially the PMT and in monochromator systems the monochromator grating. The relative efficiency varies according to the wavelength and has an effect on the spectra. If these instrument dependent effects are not eliminated from the spectra then the spectra does not precisely represent the true chemical spectra and are referred to as technical spectra. The Varioskan Flash has a unique spectral correction feature which can be used to correct the shift of the technical spectra caused by the effects of the instrument’s detection optics.

Conclusions• Withthisfullyautomaticgainselectionitispossible

to use optimal gain voltage to measure each sample individually.

• Whenahighemissionsignalsampleismeasured,alowgain voltage is used to ensure sufficient measurement range without the detector saturation.

• Similarly,whenalowemissionsignalsampleismeasured, the high gain voltage is used to guarantee good signal to noise ratio and therefore good assay sensitivity.

• Automaticgaincalibrationandselectionmakesitpossibleto obtain simultaneously both high assay sensitivity and dynamic range sufficient for any fluorometric application.

• Spectralcorrectionfeatureisneededtomeasureinstrument independent chemical spectra and can be valuable in assay optimisation when small changes in wavelengths can improve the assay performance considerably.

Above: Thermo Scientific Varioskan Flash® spectral scanning reader


Bio-Innovation30

Electronic Pipettes for Accurate and Efficient Preparation of Standard Curves

Introduction Accurate preparation and measurement of a standard curve is essential for the precise determination of analyte concentration. Because preparing standard curves is a daily routine in many biology and chemistry laboratories, pipette selection for this task is critical.

This application note discusses the use of a Thermo Scientific Finnpipette Novus Electronic Pipette for the preparation of standard curves. The pipette’s performance was compared to a manual Thermo Scientific Finnpipette model to determine if there were appreciable differences in pipetting speed and accuracy during curve preparation.

Testing with the Novus pipette was conducted in two different ways. First, curves were prepared using the “Forward” (standard) pipetting function, then curves were prepared using the “Dilution & Mix” function. The time required to prepare each dilution series was recorded (Fig. 3). The protein concentration was determined with the OPA-solution that allows for fast quantitation of protein or peptide in solution. This solution reacts with primary amino acids, resulting in highly fluorescent isoindole derivatives.

100μl of the BSA dilutions was pipetted as three replicates into black Microtiter® microplates, then 100μL of OPA solution was added. The microplates were centrifuged at 2,000rpm for 2 minutes. Fluorescence was measured using the Varioskan® multimode microplate reader. Standard curves were prepared on three different days to verify repeatability. Linear lines were used to connect the measurement points, then R2- values were calculated. An R2-value is a measure of the exactness of the linear regression; “1” represents a perfect fit between the data and the line connecting them, while “0” represents no statistical correlation between the data and the line.

Results The BSA standard curves prepared with a Novus electronic pipette and those prepared with a Finnpipette manual pipette are shown in Figures 1 and 2, respectively. The standard curves prepared with the electronic pipette using the forward pipetting function were similar to those prepared with the Dilute and Mix mode. While the R2-values for all standard curves were > 0.99, indicating a good correlation between the measurement points to a linear line, the curves prepared with the Novus electronic pipette were more convergent than those prepared with the manual pipette. The time required to prepare the dilution series ranged from 4.5 to 6.5 minutes (Fig. 3). The Novus electronic pipette was 20–30% faster than the manual pipette, an advantage especially important when using sensitive analytes that can degrade or oxidise when dispensed slowly. The Novus

electronic pipette’s Dilute and Mix mode enabled faster preparation of standard curves and also resulted in a more efficient workflow. The need for a vortexer was eliminated, which reduces the risk of repetitive strain injuries (RSI).

Conclusion The versatile Finnpipette Novus Electronic Pipette was shown to be an excellent tool for the preparation of standard curves. The electronic pipette demonstrated the ability to perform more accurate curves in 20–30% less time than with a manual pipette. Curve accuracy was due in part to the electronic pipette’s index finger operation, which helps to maintain optimal pipette positioning, and its motorised piston movement, which ensures constant pipetting speed.

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LHC Research Group, Thermo Fisher Scientific, Vantaa, Finland

Novus Electronic pipette

Finnpipette Manual pipette


thermofisher.com

Bio-Innovation 31

Introduction The promise of pluripotent stem cells lies in their ability to form any cell or tissue in the body. However, this promise requires a stable and reproducible method to grow the cells. Current methods rely on feeder cells or extracellular matrix proteins to cover the cultureware growth surface, and either manual selection or enzymatic dissociation in cell passaging and harvesting. This technical note describes a novel and simple method to grow pluripotent stem cells without the use of feeder cells or extracellular matrix proteins.

Methods Human ESC cultivation Cells. Passage-49 human ESC (H1 line from WiCELL, USA) were maintained in mouse embryonic fibroblast (MEF)-conditioned medium on Nunclon™ surface (Thermo Fisher Scientific, Denmark) coated with a 1:30 dilution of growth-factor reduced Matrigel™ (Becton Dickinson, USA). Cells were dissociated from the surface for passage by treatment with 1 mg/ mL collagenase, and then seeded onto Nunclon™ Vita™ surface with or without Rho-kinase inhibitor in the medium, as described below.

Cultivation without Rhokinase inhibition. H1 ESC were grown for 4 passages in MEF-conditioned medium on Nunclon Vita surface. Cells were dissociated from the surface for passage by treatment with 1 mg/ mL collagenase. Normal passage time for H1 ESC was 3-4 days on Matrigel™. However, cells plated on the Nunclon Vita surface took 7 days of culturing before they were ready for passage, and a spontaneous decrease in growth rate over the passages was observed.

Cultivation with Rho-kinase inhibition. H1 ESC were grown in MEF-conditioned medium supplemented with Rho-kinase inhibitor, Y-27632 (10 μM unless otherwise indicated; Sigma-Aldrich, USA). Cells were dissociated from the surface for passage by treatment with 1 mg/ mL collagenase. Cells plated in medium with 10 μM Y-27632 on the Nunclon Vita surface were ready for passage 4 days after plating. Cells were grown for the number of passages indicated.

Human ESC characterisation Colony presence and morphology were determined using phase contrast microscopy, and by the naked eye after staining colonies with 0.5% crystal violet. Pluripotency was determined by the presence of pluripotency markers through the use of qRT-PCR for gene expression, flow cytometry for cell surface marker expression, and immunofluorescence for cell surface and nuclear proteins.

Karyotypic stability was determined by cytogenetic analysis of 20 G-banded metaphase cells, and by fluorescent in situ

Feeder Cell- and Extracellular Matrix-Free Cultivation of Human Pluripotent Stem Cells

hybridisation (FISH) on 200 interphase nuclei using probes for the ETV6 BAP (TEL) gene located on chromosome 12 and for chromosome 17 centromere. Ability to form embryoid bodies was determined by growing ESC in a low-binding plate for 10 days in DMEM/F12 containing 10% FBS.

Conclusions The Nunclon Vita surface supported feeder cell- and extracellular matrix-free attachment, colony formation and growth of human ESC: • Forafewpassagesinmediumconditionedbymouse

embryonic fibroblasts • Forseveralpassagesinmediumconditionedbymouse

embryonic fibroblasts and supplemented with Rho-kinase inhibitor Y-27632

Human ESC grown 11 passages on the Nunclon Vita surface in medium with Y-27632 had normal karyotype, expressed pluripotency markers, and could be differentiated into embryoid bodies. Human ESC could be passaged without the use of enzymes or manual selection by withdrawing the Rho-kinase inhibitor from the culture in order to lift the cells from the Nunclon Vita surface, followed by re-plating cells in the presence of the Rho-kinase inhibitor.

Figure 1. Phase-contrast micrographs of human ESC passaged twice on 1:30 dilution of Matrigel™ (A), a standard tissue culturetreated surface (B), or the Nunclon Vita surface (C).

Figure 2. Dose-response effect of Rho-kinase inhibitor on the attachment of human ESC to the Nunclon Vita surface. Y-27632 was added to the cultures at a specified concentration (0, 1, 2, 4, or 10 μM) at seeding. The cells were then maintained from day 2 onward in media containing 10 μM Y-27632 with daily media changes for five days after which cells were stained with crystal violet.

Figure 3. Detachment of human ESC from the Nunclon Vita surface upon withdrawal of Rho-kinase inhibitor. Cells were seeded and maintained for 4 days in medium containing 10 μM Y-27632 (left well) or Y-27632 was removed from medium for 24 hours on the 3rd day (right well). After 4 days in culture, the cells were stained with crystal violet.

Human ESC can be passaged a few times on the Nunclon

Vita surface before the growth rate spontaneously declines

the decline in growth rate of human ESC on the Nunclon

Vita surface is not observed if the culture medium is

supplemented with Rho-kinase inhibitor Y-27632

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Application

Global Research Group, Thermo Fisher Scientific, Nunc, Roskilde, Denmark

To request a sample or for a complete copy of this article, please email: [email protected]

Bio-Innovation32

Rapid and reproducible DNA isolation from 1mL of whole blood Sini Suomalainen, Maija Partanen, Ritva Javanainen, Virpi Puro and Arja Lamberg Thermo Fisher Scientific Oy, Vantaa, Finland

Application

Sample preparation is often a limiting step for genomics and proteomics studies. Rapid and efficient isolation of nucleic acids, proteins and cells from complex biological matrices is needed to get high quality starting material for various experiments. Thermo Scientific KingFisher is a magnetic bead based automated purification system that provides a quick and easy solution to achieve high quality and reproducible results in purification of nucleic acids, proteins and cells with minimal hands-on time. This technology is based on magnetic rods which move particles through the various purification phases – binding, mixing, washing and elution. The KingFisher® is an open and flexible system, allowing the use of any available magnetic particle based purification kit suitable for the application.

This application note shows the benefits of KingFisher Flex by using genomic DNA isolation from blood as an example.

Materials and Methods -for full materials and method, please request full application note AP-MIB-KFFLEX-0508

The gDNA isolation was done for the cross-contamination test from 20mL whole blood pool by using 1mL of blood for each positive sample well. 1mL of TE buffer (pH 8.0) was used as negative samples. The test was done on the KingFisher Flex 24 format. The positive and negative samples were pipetted to every other well of the 24-well plate. The DNA extraction was done according to kit instructions. The DNA cross-contamination between wells was tested by performing 50µL PCR for all eluates of the plate with CD19 primers that produce 720 bp PCR product from human chromosome 16.

Blood samples were pooled to 20mL and 6 samples of 1mL from the pool were taken for DNA isolation with KingFisher Flex 24 format according to the 5-fold volumes of each manufacturer’s instructions. The elution volume was 500µL for all samples. The absorbances of 260, 280 and 320nm were measured from 1:5 dilutions of the eluates in a spectrophotometer and the quantity of DNA was calculated from the A260-A320 value.

The DNA quality was controlled by the (A260-A320)/ (A280-A320) ratio and 5µL of the samples were run on 1% agarose gel with 100 V. The spectra from 230nm to 320nm were measured with Thermo Scientific Varioskan.

Results In the cross-contamination test the gDNA yield was in average of 75.0 ng/µL with the ratio of OD 1.8 [(A260-A320)/(A280-A320)]. The PCR results from the cross-contamination

test show that all the negative samples from every other well produce no PCR product, meaning there wasn’t any cross-contamination from the neighbour well.

For the magnetic particle kit comparison the results between kits were at similar levels. All the kits produced good quality DNA with the OD ratio A260/A280 of 1.8-1.9. The average total quantity of DNA isolated from 1mL of blood was over 40µg with all the kits used and the agarose gel showed that the gDNA was intact.

In the isolation method a comparison of both methods (spin column & bead) used in the experiment produced equal amount of gDNA. The isolation of 24 blood samples with KingFisher Flex instrument took a total of 1 hour 18 min and filling the plates (hands on) took 22 min of that time. For the spin column method the total time was 2 hours 17 min with hands on time of 1 hour 42 min.

Discussion These experiments show that KingFisher Flex 24 is an excellent tool for high volume purification with different magnetic particle kits. KingFisher Flex is a truly open platform that produces high quality reproducible results with all the tested magnetic particle based kits and the results are comparable to the spin column method. With the Thermo Scientific BindIt Software it is easy to design protocols for magnetic particle kits from different kit manufacturers.

The data presented is based on isolating genomic DNA from blood. The amount of DNA is dependent on the amount of the white blood cells in the sample, therefore the blood samples are pooled for kit comparison experiments to minimise the variation due to the starting material.

Conclusions • KingFisherFlexutilisesafastandreproducible

automated method to purify DNA from high volume blood samples

• From1mLofbloodthetotalDNAyieldis>40µgwith all the kits used

• KingFisherFlexistheonlyopenplatformthatcanbeused with different magnetic particle kits resulting in

high quality DNA with all the kits • KingFisherFlexminimiseshands-onworkin

the laboratory and therefore saves time and increases productivity

• KingFisherFlexusesuniquepatentedtechnology which is ideal for a wide variety of samples For further details, please

email: [email protected]

Bio-Innovation

Peter Esser, Senior Scientist, and Louise Gjelstrup, Laboratory Technician Thermo Fisher Scientific Laboratories, Roskilde, Denmark

33

It is a well known fact that temperature gradients and vibrations during cell settlement in cell culture flasks and plates may cause uneven cell distribution patterns on the growth surfaces [1, 2]. Therefore, all ingredients assembled should be left in absolute tranquillity (i.e. no temperature gradients, no vibrations, and no ventilation) during cell settlement. This is most easily accomplished by pre-incubation of the seeded culture at room temperature (RT).

The significance of the evaporation reservoir in the Thermo Scientific Nunc Edge Plate in relation to the uneven cell distribution was investigated with either 200 or 100µL MDCK cell suspension per well according to the following 4-plate test set-up distinguishing four different situations framed in red:

ResultsWith 200µL of cell suspension per well it was seen that without pre-incubation (plates 1 and 3) outward “half-moon” cell accumulations occur in the edge wells, but to a lesser degree in the plate with the reservoir filled (plate 1) compared to the plate with an empty reservoir (plate 3).

Intrawell Cell Distribution in MicroWell Edge Plates A

pplication

In the latter case, with an empty reservoir and no pre-incubation, additional patterns occur in the edge wells, which may stem from incubator vibrations. However, both phenomena are eliminated by pre-incubation (plates 2 and 4). It is possible therefore, that the reservoir content, may to some extent act as a “buffer” against uneven cell distribution in the (edge) wells but has no significance if pre-incubation is employed.The results with 100µL cell suspension per well, showed that the edge effects are largely absent, thus indicating a volume-dependent reverse effect. This may be explained by the shorter settling distance and time, making the cell distribution less sensitive to thermal disturbances in the wells.

Conclusion In conclusion, the recommendation of tranquil pre-incubation during cell settlement is maintained for the Nunc Edge Plates on the condition that the reservoir content has the same temperature as the other ingredients during the pre-incubation. However, the extent of thermal disturbance is also dependent on cell type, thus with MRC5 cells we observed almost no such effect (results not shown). With suspension volumes reduced to 100µL per well, pre-incubation may be unnecessary, but users may normally avoid smaller volumes because evaporation would be more critical. This condition could be eliminated by using Edge Plates with the reservoir filled.

For a copy of the complete application note, please email [email protected]

Plate Number 1 2 3 4

Conditions

Plate RT RT RT RT

Cell Suspension RT RT RT RT

Reservoir RT Water* RT Water* Empty Empty

Pre-incubation none 2hrs @ RT none 2hrs @ RT

Incubation** 37°C 37°C 37°C 37°C

References: 1. Nielsen V. and Esser P. Incubator Shelf “Images” in Monolayer Culture. Nunc Bulletin No. 3, 2nd Ed. 1997.

2. Nielsen V. Vibration Patterns in Tissue Culture Vessels. Nunc Bulletin No. 2, 2nd Ed. 1997.

3. Lundholt B. K. et al. A Simple Technique for Reducing Edge Effect in Cell-Based Assays. Journal of Biomolecular Screening 8(5), 2003.

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To request a sample or for a complete copy of this article, please email: [email protected]

Bio-Innovation34

The Importance of Optimising the DNA Purification Protocol in Magnetic Particle-Based Systems

Article

Magnetic particle-based techniques are widely used in many diverse biological applications, offering a relatively inexpensive technology and subjecting samples to very little mechanical stress. Although referred to as “magnetic,” the majority of the particles used are paramagnetic or superparamagnetic, meaning that the beads only exhibit magnetic properties in the presence of a magnetic field, with no residual magnetism once removed. A wide range of bioreactive molecules can be adsorbed or coupled to the bead polymer surface and used in the separation of biological materials such as cells, proteins, DNA, and RNA. Paramagnetic beads are particularly suitable for automated procedures because the instrumentation exists to easily mix, incubate, and separate the particles in 96-well plates without columns or centrifugation.

Increasing range of applications Magnetic particles are increasingly being used as carriers for binding proteins and enzymes in proteomics applications, where the immobilised biomolecules can be used directly for a bioassay or as affinity ligands to capture or modify target molecules or cells. In addition to whole cell isolation, even cell organelles can be selectively separated using magnetic particles. Immunomagnetic cell isolation and separation methods are also proving useful in cell sorting applications, for example, in the isolation of rare progenitor cells from human cord blood. Magnetic particle separation has proven most invaluable, however, for sample preparation in drug discovery and genomics applications, including high-throughput genome isolation for sequencing or PCR amplification for downstream processing such as single nucleotide polymorphism (SNP) analysis or expression profiling. The particles offer many advantages, including reduced reagent costs, simplified procedures, and reduced time needed to achieve good yields of high-purity nucleic acids.

Flexible solutions for nucleic acid purificationMagnetic particle-based kits offer excellent performance for the purification of DNA and RNA. The range of starting material for nucleic acid research can be broad—from 5mL of blood for routine testing to very small samples, such as those obtained from forensic surface and contact swabs, hair, and cigarette butts. The technique enables the isolation of DNA or RNA in sufficient quantities from all kinds of material sources such as serum, blood, bacteria, cell culture, tissue, food, soil, or faeces. Kits can also be used to purify DNA from several plant species and sources. Use of magnetic bead-based separation technology ensures that the resulting DNA and RNA are free of protein, nucleases, and other contaminants, ensuring that purified nucleic acid samples are ready to use in downstream applications.

Speed and sample purity continue to be a major concern to genomics and proteomics researchers. The sheer volume of work involved in genome and protein mapping means that processing speed is essential. Unsatisfactory results on even the most sophisticated analysers are increasingly linked to poor initial sample quality. The variety of samples being used, however—from rare cell types and mRNA transcripts to increasingly large and delicate protein complexes—presents a real challenge.

Automating such purification procedures ensures impurities are removed rapidly, efficiently, and consistently; an open and flexible automation system will enable the user to choose any available magnetic particle-based purification kit suitable for the application. Predefined application protocols are available for many existing magnetic particle based methods, and it is easy to customise existing protocols or create entirely new ones using appropriate intuitive software. In addition, it is important to

select a flexible processor that offers high throughput and a wide range of processing volumes, such as the KingFisher® Flex.

The purification protocol is created with the associated Thermo Scientific BindIt 3.1 software, which allows the user to optimise the purification conditions taking into consideration the sample type and particles used in the extraction kit. Several different magnetic heads and plate formats are available. This study used 96-deep-well and 24-well magnetic heads & tip combs.

Optimisation of the DNA purification protocolThe DNA purification protocol typically consists of cell lysis/DNA binding, several washing steps, and the DNA elution. All of these purification steps were optimised for both 96- and 24-deep-well formats. In the 96-well format, the DNA extraction was performed from 200μL blood or ~6μg pure calf thymus DNA, and in the 24-well format, from 1mL blood or ~35μg calf thymus DNA. Blood DNA purification kits from two vendors were used. The absorbances of the eluents were measured, the DNA yield was calculated based on the measured 260-nm absorbance, and the quality was determined by analysing the 260/280 nm ratio. The quality of DNA was also tested by end-point polymerase chain reaction (PCR) to control the presence of PCR inhibitors in the eluent. The protocols were optimised by changing one variable at a time, e.g., the mixing speed of one step was optimised by alternating the available mixing speeds while the rest of the protocol remained unchangeable.

Effects of different mixing speedsThe different mixing speeds had a strong effect on the DNA purification. For DNA purification, it is important to mix the solutions efficiently to reach optimal binding of the DNA and the bead. The faster mixing speeds increased DNA yields for DNA

Thermo Fisher Scientific, Ratastie 2, P.O. Box 100, Vantaa, FIN-01621, Finland

Bio-Innovation 35

Figure 2 Effects of the wash step mixing speed on the final DNA quality. PCR products of the elutions. a)Slow, b) medium, c) fast.

purification from blood. When one variable at a time (binding, washing, and elution) was tested, the faster mixing speeds for all steps resulted in a better yield (Table 1). The mixing speed makes the greatest difference to the results with the 24-well format, due to the shape of the tip and the well. Powerful washing is essential for the high purity of the DNA. The protocol parameters in the binding and elution steps affected, primarily, the total DNA yield. In the washing steps, the slow mixing had an effect on the DNA quality. The purified DNA was analysed with PCR, and the results show the remaining impurities affecting the secondary applications (Figure 2).

Table 1 shows the effects of different mixing speeds for binding and elution steps and the effect of the heating and elution volume on the final DNA yield. Results of the slow mixing speed indicate the impurities in the elution and inhibition of the enzymatic reaction due to inefficient washes. Usually, slower mixing speeds are preferred during elution due to DNA degradation, but, according to this study, fast mixing in the elution results in the best quantity and quality DNA. The slow mixing speed is useful for the heated steps of the nucleic acid purification. It should be noted, however, that the same optimisation is not directly applicable to RNA and protein purification, for example. Half mix, medium, or slow mixing speeds are recommended for more sensitive bead–biomolecule complexes.

ConclusionMagnetic separation technology is increasingly becoming an integral part of the laboratory in this post genomic era, finding a role in biomedical, clinical, and cell sorting applications and proving an invaluable part of the process in proteomics, drug discovery, and genomics. The range of magnetic particles now available enables routine separation and purification of biological materials such as cells, proteins, DNA, and RNA. Automation of this technology offers many advantages in terms of increased efficiency, throughput, reliability, consistency, reduced costs, and enhanced yields. All of the purification steps require beads to separate effectively, and

this study found that mixing speeds are significant for optimal binding, efficient washing, and active elution.By simple optimisation of the magnetic particle-based nucleic acid purification protocol, it is possible to provide a rapid and reliable method for DNA isolation and achieve a good yield of high-quality DNA. Use of an open-platform automation system and intuitive software, which is easy to modify, enables the user to take into consideration differing sample types and particles. Rapid and reliable separation methods and easy adjustment of isolation protocols should allow complete flexibility of use for all future applications.

Table 1 Optimising binding and elution of the DNA purification protocol

A B C

Slow Medium Fast


Bio-Innovation36

the thermo Scientific Pierce Fast Western Blot

Kits accelerate the Western blotting process

with streamlined protocols and optimised

reagents that provide accuracy, sensitivity and

reliability. the new kits reduce hands-on and

overall blotting time to approximately one hour

using any of the thermo Scientific SuperSignal

Chemiluminescent Substrates.

Highlights: • Reliable–alltheperformanceyouexpectfromour

SuperSignal Substrates • Convenient–noexpensivehardwareneeded;eliminates

membrane clogging issues and excessive waste • Simple–optimisedprotocolmakesWesternblotanalysis

easier than ever • Economical–noexpensiveconsumablesorextra

equipment required • Stable–kitisstableforoneyearat4°C

After transferring proteins to a membrane, traditionalWestern blotting procedures can take 4 hours or more. Our kits provide all the reagents necessary to complete a Western blot in ~60 minutes (Figure 1). The protocol is easy and requires no additional optimisation. Simply use a mouse or rabbit primary antibody at the same concentration used for classic Western blot with SuperSignal Substrate. Because our fast system is reagent-based, there is no investment in equipment or costly consumables to purchase. Our specially formulated blocking buffers and detection antibodies accelerate Western blotting by minimising incubation times without sacrificing sensitivity. Additionally, the blots can be stripped and reprobed, thus saving even more valuable time.

New Western blotting kits for fast results

The Pierce Fast Western Blot Kits contain everything required post transfer to obtain your data quickly and easily. The protocol is simple with the flexibility to run one or numerous blots in approximately one hour. To demonstrate the effectiveness of the fast system, we compared blots that were prepared using the classic Western blotting protocol to results obtained using the Pierce Fast Western Blot Kits. The three SuperSignal Substrates (West Pico, West Dura and West Femto) were evaluated

Methods: Classic Western blot protocol: Membranes were blocked with 5% milk in Tris-buffered saline with Tween*-20 (TBST) for one hour and then probed with primary antibody for one hour. The membranes were washed with TBST three times for 5 minutes each. Horseradish peroxidase conjugated to goat anti-mouse or goat anti-rabbit antibody was diluted in 5% milk TBST (20 ng/mL for SuperSignal West Pico and Dura Substrates and 4 ng/mL for SuperSignal West Femto Substrate). The blots were incubated for 1 hour in secondary antibody solution. The membranes were washed with TBST six times for 5 minutes each and developed using the appropriate SuperSignal Substrate.

Total protocol time, post-transfer to substrate incubation, was approximately 4 hours. Fast Western blot protocol: Membranes were incubated for 30 minutes in primary antibody diluted in 10mL of Antibody Diluent. The membranes were incubated for 10 minutes with 1mL of Optimised Mouse or Rabbit HRP Reagent in 9mL of Antibody Diluent. The membranes were washed with three rapid water rinses followed by three 5-minute washes in Wash Buffer. An additional water rinse was performed after each wash for blots developed with SuperSignal West Femto Substrate. The membranes were developed using the appropriate SuperSignal Substrate. Total protocol time, post-transfer to substrate incubation, was approximately 1 hour.

BlockMembrane

PrimaryIncubation

Washes SecondaryIncubation

Washes

PrimaryIncubation

HRP Reagent

SuperSignal

Substrate

Incubationin Substrate

+ + = 3 hours, 45 minutes

Classic Western

Fast Western

Washes

55 minutes

1:0000

0:3000

1:0000 0:1500 1:0000 0:3000

0:1000 0:1500

0:0500 SuperSignal

Substrate

Figure 1. Complete Western blots in ~1

hour using any Thermo Scientific Pierce Fast

Western Blot Kit, SuperSignal Substrate.

For a complete copy of this article, please

email: [email protected]

1 hour

Complete Western blots in

Application

Thermo Scientific Pierce. 3747 N. Meridian Road, Rockford, IL 61105-0117, USA

Bio-Innovation 37

Direct PCR from plant tissue without DNA purification using Finnzymes’ Phire® Plant Direct

Application

Finnzymes’ Direct PCR approach allows amplification of DNA directly from different types of starting materials without prior DNA purification. The Phire® Plant Direct PCR Kit is a complete set of optimised reagents and sampling tools developed especially for plant PCR. Based on unique protein fusion technology, this kit combines a specially engineered DNA polymerase with buffers that have been optimised for PCR using leaves, seeds and flowers as a template.

Introduction DNA extraction and purification from various plant sources requires large amounts of sample material, and adds time and expense to PCR protocols, as well as increasing the risk of cross contamination and human error. The Phire Plant Direct PCR Kit is designed especially for plant samples; pieces of leaves, seeds and flowers, or punches from FTA® Cards may be added directly to the PCR reaction with excellent results. In addition, only a very small sample (0.5mm leaf punch) is enough for the assay. For situations where a larger sample of tissue is used, or when multiple PCR reactions are to be run from a single sample, we have developed a suitable dilution protocol employing a brief preincubation in an optimised dilution buffer. An aliquot of the sample in buffer can be used directly in PCR reactions. For the direct protocol, the Harris Uni-Core™ puncher was used to obtain consistently-sized samples. The Phire Plant Direct PCR Kit also includes a pair of universal control primers that work with a large variety of plant species. All protocols were run on the Piko Thermal Cycler with UTW plates and tubes. This integrated system allows consistent results to be obtained in the shortest possible times.

MethodsFor Direct PCR protocols, a 0.5mm disc of tissue was placed directly in the reaction vessel, along with 20-50μl of PCR mix.Aninitial5-minuteincubationstepat98°Cwasincludedto release DNA, followed by an optimised fast protocol. For samples on storage cards, direct PCR with 1mm punches were used with reaction volume of 50μl. For dilution protocols, a 2mm disc of tissue was crushed in 20–50μl of the dilution buffer and centrifuged. Subsequently, the supernatant (0.5 to 2μl) was used as template for PCR.

For a complete copy of this article, please email: [email protected]

Figure 1. Direct PCR was performed using a 0.5mm punch from leaves of maize, tomato and Arabidopsis plants in a 20μl PCR reactions. Mitochondrial (maize) or genomic (tomato and Arabidopsis) targets of 1.8 to 3.5 kb were amplified using the Phire Plant Direct PCR Kit (3-step protocol, 40 cycles, as recommended in the kit manual. M size marker, + purified DNA control, − negative control with no template DNA.

Figure 2. Direct PCR of a 1.8 kb fragment of the genomic DNA from tomato seeds. Fragments of tomato seeds were dehulled and various sized fragments were placed directly into a 20μl PCR reaction. For the dilution protocol, a larger seed fragment was placed in 20μl of Dilution Buffer and incubated for 3 minutes at room temperature. A 0.5μl aliquot of the supernatant was used in a 20μl PCR reaction directly, or diluted 10 or 100 fold, and then used as template. M size marker, + purified DNA control, − negative control with no template DNA.

Figure 3. Punches (1mm) from FTA Gene Saver Cards were placed directly in 50μl PCR reactions and amplified using universal plant mitochondria specific primers, producing 1.0- 1.8 kb products.

Figure 4. Fungal DNA was amplified from the roots of spruce trees (0.5mm to 1mm in length) in a 50μl PCR reactions containing primers specific for a 1.0kb target of the fungus Cenococcum. Product of correct size was cleanly amplified over a range of input sample quantities. M size marker, − negative control with no template DNA.

1 = Maize (husk)2 = Snap Dragon (petal)3 = Dalia Purple (petal)4 = Dalia Pink (petal)5 = Pea (leaf)6 = Bean (leaf)7 = Tomato (leaf)8 = Dalia Red (petal)9 = Dalia Yellow (petal)10 = Negative control11 = Arabidopsis (leaf)

Figure 1. Direct PCR form 0.5mm leaf discs

Figure 2. Direct PCR from tomato seeds

Figure 3. Direct PCR from plant material stored on FTA cards

Figure 4. Amplification of fungal DNA from plant roots

Conclusions 1. The unique properties of the polymerase and optimised reagents of the Phire Plant Direct PCR Kit enable Direct PCR protocols not feasible with conventional enzymes2. Only a very small sample is required for robust Direct PCR protocols. The Harris Uni-Core puncher is an excellent tool to obtain consistent small samples. 3. The dilution protocol is especially convenient when multiple PCR reactions are performed from a single sample, or when larger samples (e.g. 2mm punch discs) are to be used without further sample manipulation. Finnzymes has developed an optimised dilution buffer for plant samples. 4. The Phire Plant Direct PCR Kit may also be used to detect some species of fungi. Updated list of tested plants available at www.finnzymes.com/directpcr

Results

Pak Yang Chum and Chas André, Finnzymes Oy, Keilaranta 16 A, 02150 Espoo Finland

Bio-Innovation38

Five brands of filter tips were tested for their ability to prevent carryover contamination by over pipetting PCR* product through the filters and running the recovered sample on 1% agarose gel. Filter protection was shown to be nonexistent in Oxford, LabCon, USA Scientific and Continental Laboratory Products, Inc., (CLP) filter tips. Only ART® tips manufactured by Molecular Bio-Products, Inc., completely blocked the passage of liquids and DNA. Of the five filter tips tested, only ART® tips provided complete and consistent protection against pipettor contamination.

Introduction Filter protected pipet tips are specifically designed to prevent carryover contamination from liquids and aerosols that may come in contact with the pipettor. For example, PCR product contains DNA molecules which will spread through direct contact with the pipettor barrel, or by aerosols generated by the action of pipetting. The resulting contamination can adversely affect subsequent reactions and lead to inaccurate results. This potential for error illustrates the importance of an effective filter barrier.

Objective Various filter technologies have been developed, each claiming to prevent the passage of DNA molecules to the pipettor. The seemingly subtle differences between filters will result in error prone PCR because not all filter tips are capable of providing consistent protection against pipettor contamination. Therefore, this study was designed to test the true effectiveness of filtered pipet tips from Oxford, LabCon, CLP, USAScientific and ART® tips from Molecular BioProducts, inc.

Materials And MethodsFive conventional PCR reactions were carried out in HotStart 50® storage & reaction tubes using primers for mitochondrial D-loop DNA at 0.2 mM concentration & template consisting of 0.02 µg total human liver DNA (Life Technologies). MgCl2 concentration was 2mM, was 2mM, dNTP concentrations were 2mM and 1X PCR buffer was used. Thirtythermalcyclesconsistedof56°Cfor30seconds, 72°Cfor60secondsand94°Cfor60seconds.Eachfilter tip was then used to intentionally overdraw 20µL of PCR product using a pipettor deliberately set at 200µL. 15µL of any liquid that passed through the filters was then collected and run on a 1% agarose gel paired with 15µL of the same product taken from the respective original reaction tubes.

Results Only ART’s patented filter prevented fluid from passing beyond the filter. LabCon and USA filters were penetrated almost immediately and both Oxford and CLP filters failed within two - three seconds. Every filter that failed allowed

Keep it Clean with ART® self sealing barrier tips

18-20µL of liquid to pass through freely, creating the potential for pipettor contamination. The 15µL aliquots recovered from behind the filters and the positive controls consisting of “unfiltered” samples were run on a 1% agarose gel containing ethidium bromide at V=100 for 25 minutes. (Fig.1)

Lanes 1, 3, 5 and 7 are aliquots recovered from behind the filters of Oxford, LabCon, USA and CLP tips respectively. Lanes 2, 4, 6 and 8 are “unfiltered” control aliquots from the respective original microfuge tubes.No visible or quantifiable differences existed between the “filtered” and control aliquots indicating that all but ART filter tips permitted liquid sample containing DNA to pass freely through the filter. It was therefore concluded that ART filter tips have the only effective barriers that protect against carryover contamination.

DiscussionFilter protected tips have been designed to specifically prevent carryover contamination. It is clear from this study, however, that not all filter technology is as effective as that which is patented by Molecular Bio-Products Inc.Additional evidence of ART’s effectiveness is discussed in the article “Use of Filtered Pipet Tips to Elute DNA from Agarose Gels” BioTechniques, June 1995. The article describes the process of drawing DNA through the filters of Costar and Bio-Rad filter tips. DNA passes through the filters freely “without apparent effect to its integrity.” It was further determined that “ART tips, which are designed to seal when exposed to liquids, could not be used for this method of eluting DNA.” ART tips permitted no visible or measurable penetration of DNA and are the only filters proven to offer complete protection against pipettor contamination.*PCR process is covered

by patents owned by Hoffmann- La Roche,

Inc. ART is a registered trademark of MBP and is protected by

U.S. Patent Number 5,156,811; HotStart 50 is

a registered trademark of MBP; MBP is a

registered trademark of Molecular BioProducts,

Inc., San Diego, CA. ©MBP, 2000.

Filter protected pipet tips specifically designed to prevent carryover contamination from liquids and aerosols that may come in contact with the pipettor

Pipette Tip with ART Barrier

Pipette Tip with Ordinary Filter

Self-sealing barrier tips block contamination

Other filtered tips allow contamination to pass through

1 2 3 4 5 6 7 8Lanes


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Solaris Q & A

What are Supernucleotides and what function do they perform in my Solaris qPCR Gene Expression Assay? Supernucleotides (Superbases) are functionally equivalent chemically modified versions of native nucleotides. These specialised nucleotides have a higher melting temperature (Tm) and are used to precisely adjust the overall Tm of the probe/primer sequence to which they are added. In this way they function to accurately maintain what is commonly referred to as “universal thermal cycling conditions” as well as to mitigate any unfavorable secondary structure within the sequence.

How can I be sure that my Solaris Assay is target specific? The Solaris design algorithm performs a BLAST search on transcript, genomic and pseudogene databases to ensure all assays are specific.

What is an MGB moiety and what function does it perform in my Solaris qPCR Gene Expression Assay? A minor groove binder (MGB) moiety, which is attached to the 5’ end of a Solaris probe, increases its melting temperature, thereby allowing shorter probes to be designed. This increases design flexibility and gives improved assay performance. Furthermore, the MGB moiety, in conjunction with Superbases, gives the same Tm (60ºC) for every Solaris probe. This means the same thermal cycling conditions can be used for all Solaris Assays.

Can I perform a faster protocol using Solaris Assays? Most Solaris assays perform very well using a faster protocol, provided they are used in conjunction with a master mix that promotes efficient amplification under these conditions.

What is the stability of the Solaris qPCR Gene Expression Assays? The shelf life is at least 2 years when stored at -20ºC.

What is the difference between “Inventoried” & “Made to Order” Solaris qPCR Gene Expression Assays? Inventoried Assays are those commonly studied gene targets that have already been synthesised and placed into inventory. The delivery time for inventoried assays is 3-6 days after the order is received (direct countries only). Made to Order Assays are those less commonly studied gene targets that have already been designed but not yet synthesised. The delivery time for these assays is 5-12 days after the order is received (direct countries only).

What is meant by “universal thermal cycling conditions” when referring to my Solaris qPCR Gene Expression Assay? This refers to the use of identical thermal cycling conditions for all Solaris qPCR Gene Expression Assays, irrespective of the target gene.

Can I use my Solaris qPCR Gene Expression Assay to detect specific splice variants of a gene target? The Solaris qPCR Gene Expression Assays have been designed to detect all known variants of a given gene target, thereby giving highly accurate quantification results. They cannot be used to detect a specific splice variant.

Will my Solaris qPCR Gene Expression Assay detect genomic DNA (gDNA) if it is designed within a single exon? Solaris assays that are designed within a single exon may detect genomic DNA if it is present. This design information is provided for all Solaris Assays. Con-sequently, gDNA should be completely removed prior to the RT step.

Following the release of Solaris qPCR assays we have been inundated with questions regarding the design and performance of the product. Here are the most commonly asked questions with answers from our Tech Support team.

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Stem Cell Promise Research Brings Autograft

Revolution Closer

Vita means Life Unique energy treated polystyrene surface

that enables growth of most cell lines without using matrix coatings or feeder cellsusing matrix coatings or feeder cells

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Unique energy treated polystyrene surface that enables growth of most cell lines without

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