Integrated Imaging Solutions for Robust and Replicable Data

Transcription

1 Integrated Imaging Solutions for Robust and Replicable Data 1

2 BRC, We Have a Problem All of us in the Biomedical Research Community (BRC); academia, industry, journal publishers, funding agencies, and government. Published Data are Not Consistent with Replicated Studies Among other findings out of 67 Studies 6 out of 53 Findings showed inconsistencies between published and in-house data when replicated by researchers at Bayer1 from landmark preclinical cancer papers could be confirmed by researchers at Amgen2 Variability in Materials and Methods at Fault Among other sources... 11% 28% Lab Protocols 36% 25% Biological Reagents & Reference Materials 2 Study Design Data Analysis & Reporting 54% of Materials 4 Major Factors used in research, including antibodies, model organisms, and reagents are not uniquely identifiable in publications3 contribute to irreproducibility in preclinical research4

3 The Bottom Line Hit Hard Among other costs... $ $ 28 Billion Spent Annually on research that cannot be replicated, in the United States alone4 500,000-2 Million Per Study investment needed to replicate academic research with potential clinical applications within the pharmaceutical industry4 Your Peers Agree Among other surveys... 90% of Respondents surveyed by Nature from various scientific disciplines agreed that there is a reproducibility crisis in scientific literature5 At least 60% of Respondents in the fields of biology and medicine from Nature and American Society for Cell Biology (ASCB) surveys reported failure in replicating someone else s experiments5, 6 3

4 Change is Coming Soon to a Lab Near You December The National Science Foundation (NSF) Reported its ongoing and future activities to improve reproducibility, replicability, and robustness in funded research7 June The National Institutes of Health (NIH) Issued revisions to grant application instructions in an effort to enhance reproducibility in preclinical research8 October The Academy of Medical Sciences (UK) Developed an action plan for improving reproducibility and reliability of biomedical research9 January The Federation of American Societies For Experimental Biology (FASEB) Drafted recommendations regarding the use of mouse models and antibodies towards enhancing research reproducibility10 September The InterAcademy Partnership (IAP) for Health Issued a statement, endorsed by 46 of its member academies in Europe, Asia, the Americas, and Africa, to improve reproducibility of biomedical research11, 12 January The American Statistical Association (ASA) Provided recommendations on statistical analysis to funding agencies for supporting reproducible research13 April The German Research Foundation (DFG) Published a statement on replicability of research results and identified activities for research funding and scientific selfgovernance14 4

5 Ongoing Efforts to Enhance Reproducibility Reproducibility2020 Initiative Led by the Global Biological Standards Institute (GBSI), the initiative aims to significantly improve the quality of preclinical biological research by the year 2020, through best practices and standards15, 16 Revision of Publication Guidelines Journals groups AAAS, ASBMB, ASCB, Biomed Central, F1000, Frontiers, Nature, PLOS, Springer, and Wiley, among others, have signed up to tighten publication guidelines17 Validation of Research Materials A number of non-profit groups in partnership with academia and industry are developing standards, practices, and validation guidelines for research reagents16 Training in Rigor and Reproducibility Training programs are being developed for researchers in study design, meeting funding and publication policies, data analysis, and more15

6 Build a Robust Story Replicable Data for Publications and New Drug Applications Your research enables new ways to conquer disease: from studying disease biology and identifying targets for potential therapies in academia, to validating targets and functionalizing biomolecules into medicine in industry. Unraveling the complex interplay between biochemical pathways and understanding how therapeutic agents affect them often requires reliance on protein expression data. Results from Western blots and cell-based biochemical assays help measure therapeutic outcomes and guide preclinical development of potential drug compounds. A lot relies on accurate assessment of protein expression changes. However, variability in technique, detection methods, reagents, and data analysis could yield results that are inconclusive and irreproducible. Lifesaving therapies are being delayed, research budgets face increasing pressure, and drug development and treatment costs are rising. Improving reproducibility remains a critical cornerstone to solving each of these challenges...solutions, such as addressing errors in study design and using high quality biological reagents and reference materials, will require time, resources, and collaboration between diverse stakeholders that will be a key precursor to change. Freedman LP. The Economics of Reproducibility in Preclinical Research. PLOS Biology 13(6): e

7 Minimize Variation. Maximize Accuracy. When you need results that you can depend on, take steps to safeguard the integrity and accuracy of your data. Minimize error and variation using standardized protocols, robust detection, validated reagents, and secured data analysis software. Lower variability will give you reproducible data, in which you can be confident. Consistent and accurate results will help you make a bigger impact every step of the way from reporting data to improving healthcare outcomes.

8 Replicable Data Will Take Your Research Farther The need for consistent, replicable results has never been as pressing. In the face of fast-evolving research best practices and reporting guidelines, get two steps ahead, using technology that helps you minimize error and variability. Biomedical researchers worldwide have placed their trust in LI-COR Biosciences near-infrared (NIR) fluorescence imaging technologies for accurate detection, for more than 25 years. 10K+ Publications citing the Odyssey Imaging Systems for detection and quantification of protein expression 12+ Cyanine-based dye patents on composition of matter and methods worldwide 50+ Patents for imaging technologies worldwide 8

9 Enlist LI-COR solutions in your quest for definitive protein expression analysis. Take your studies forward with the Data Integrity Bundle. Accurate Detection Odyssey CLx and Fc imaging systems Stable Chemistry IRDye infrared dye products and reagents Reliable Analysis Image Studio data analysis software Comprehensive Learning Personalized training and educational resources Accessible Expertise Unparalleled technical support just a call away Close Collaboration End-to-end assay and validation services Learn how the Data Integrity Bundle can help you meet revised publication requirements. licor.com/pubchange 9

10 A Wide Range of Detection Strong and Faint Signals Covered When your research relies on measurement of protein expression changes, you need data that accurately represents the amount of proteins present. It is within the Linear Dynamic Range (LDR) of your Western blot assay that signal intensities detected by the imaging system are proportional to protein abundance. As the protein amount changes, the signal intensity changes proportionately, giving you accurate detection of molecular activity for quantitative analysis. Widen your detection capabilities for clear-cut answers to protein abundance. Equipped with 6+ logs of dynamic range, LI-COR Odyssey Imaging Systems provide flexibility to capture the LDR of your assays in a single acquisition toward meeting data reporting guidelines. So, confidently quantitate your results and report your findings. Quantification of gel or blot intensities must be performed with data obtained within a linear range of exposure. Instructions for Authors, Journal of Biological Chemistry Integrated intensity Background 1,000, ,000 10,000 1, pg 1.5 ng Enhance your data accuracy. The wide dynamic range of Odyssey imagers enables high-sensitivity detection without saturation. 10 R 2 = ,000 10,000 pg of Protein 10

11 Optimization Made Easy Targets and loading controls; for robust protein expression analysis, you need to integrate and correlate data from more samples than just one. Whether you are measuring target proteins in reference to total protein, housekeeping proteins, or other modified forms, you need to detect them all on the same blot, within the combined linear dynamic range of the assay. Tell the complete story in every image. The wide dynamic range of Odyssey Imaging Systems lets you easily optimize loading amounts of targets and controls within the combined linear range for accurate analysis, in line with journal recommendations. So, detect targets and internal loading controls within the linear range for consistent and reproducible results. For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used. Image Integrity: Authors and Referees, Nature Publishing Group - Nature Internal Loading Control Target Protein Combined Linear Range Linear Range Linear Range Signal Sample Loading Confidently measure protein expression changes. A wide dynamic range of detection facilitates optimization of target and internal loading control amounts within the combined linear range, for accurate analysis. 11

12 Enhanced Accuracy with NIR Quantification Amplified For measuring multiple targets with a wide range of signal intensities, efficiency and data replicability are key. Demand a solution that provides consistent data. Comparing signal intensities from high- and low-abundance targets run on separate gels/blots introduces error and variability in data analysis. When measured on the same blot, stronger signals (such as loading controls) can quickly saturate limited dynamic range detectors, before other targets are even detected. Saturated data are no longer proportional or accurate. Adapt your workflow to meet publication guidelines with multiplexing. Wide dynamic range coupled with near-infrared (NIR) fluorescence detection on Odyssey systems lends flexibility to detect multiple targets simultaneously with varying abundance. So, acquire coherent data for quantitative comparisons across all your targets and controls. The image should include all relevant controls, and controls should be run on the same blot or gel as the samples. Submission Guidelines, PLOS ONE 800 nm Channel 700 nm Channel Overlaid Images Multiplex with ease. See targets and loading controls on the same blot with multiplex detection using 700 nm and 800 nm channels. 12

13 The Smallest of Changes, Revealed Infrared detection allows you to get better sensitivity without compromising data accuracy. Laser-based illumination and the advanced optics of Odyssey imagers, coupled with low membrane autofluorescence in the NIR spectrum, deliver high signal-to-noise ratios. Low-background detection expands the range of signals that can be quantified, compared to traditional methods. A wider range of quantifiable signals makes it easier to determine if your target and internal loading control (used for normalization) can be detected in the same linear range. So, acquire data for minute changes in protein levels with high replicability. Visible Fluorescence (532 nm) Odyssey NIR Fluorescence (700 nm) Unstimulated Stimulated Unstimulated Stimulated Relative fluorescence 2,500,000 2,000,000 1,500,000 1,000, ,000 LI-COR Odyssey PVDF Nitrocellulose 532 nm 635 nm 700 nm 800 nm Get high-sensitivity detection and low membrane autofluorescence in the NIR spectrum. Phospho-ERK was detected in lysates of unstimulated or EGF stimulated A431 cells and detected at 532 nm and 700 nm, respectively, using Typhoon imager (upper left) and Odyssey imager (upper right). 13

14 Odyssey CLx Imaging System High-precision laser illumination meets high-sensitivity detection optics for accurate, replicable data capture. Odyssey CLx imaging systems are engineered to enable definitive analysis of protein expression changes. Widest dynamic range of detection that lets you see all data in a single image, without saturation Autoscan feature provides identical imaging settings from scan to scan Dual-channel fluorescence detection enables multiplexing for improved throughput and efficiency Imaging Applications Near-Infrared Fluorescence Western Blots In-Cell Western and On-Cell Western Assays Coomassie Protein Gel Documentation Near-Infrared Fluorescence Protein Arrays EMSA/Gel Shift Assays In-Gel Westerns Macroscopic Analysis of Tissue Sections

15 Specifications Image Field Size: 25 cm 25 cm, 9 mini-blots, 6 microplates Dynamic Range: 4 logs (Manual); > 6 logs (Auto) Laser Lifetime: 40,000 typical working hours 700 Channel Laser Source: Solid-state laser diode at 685 nm 800 Channel Laser Source: Solid-state laser diode at 785 nm Detectors: Silicon avalanche photodiodes Scanning Speed: 5-40 cm/s Resolution: µm Focusing Range: Microscope is adjustable 0-4 mm above the scan bed to obtain best signal-to-noise ratio Operating Conditions: C and dew point no greater than 20 C Power Requirements: Universal input range is between VAC (voltage fluctuations not to exceed 10% of the nominal voltage); 4 Amp maximum; at 120V, 0.5 Amp typical when idle; 50/60 Hz Dimensions (instrument only): 37 cm H 53 cm W 62 cm D (15" 21" 24.5") Height with hood fully open is 74 cm (29 ) Weight: 33 kg (72 lbs) The superior dynamic range and sensitivity allows me to confidently report my data. Lars Engstrom, Mirati Therapeutics, Via SelectScience

16 Odyssey Fc Imaging System High-performance laser powered excitation, reinforced by the FieldBrite XT2 core for consistent data capture. Odyssey Fc imaging systems are built to facilitate accurate quantification of protein and DNA amounts. Widest dynamic range of detection that lets you see all data in a single image, without saturation Uniform field illumination across the entire imaging area minimizes data variability Dual-channel fluorescence and chemiluminescence modes provide detection versatility Imaging Applications Near-Infrared Fluorescence and Chemiluminescence Western Blots Nucleic Acid Gel Documentation Coomassie Protein Gel Documentation Near-Infrared Fluorescence and Chemiluminescence Protein Arrays

17 Specifications Image Field Size: 10 cm 12 cm, disposable trays for DNA gel imaging available CCD Pixel Size: 6.45 µm Dynamic Range: > 6 logs Depth of Field for Best Sample Focus: 6 mm Patented FieldBrite XT2 Technology: CV < 3% across field Laser Lifetime: 20,000 hours typical 700 Channel Laser Source: Solid-state laser diode at 685 nm 800 Channel Laser Source: Solid-state laser diode at 785 nm Light Source: 600 nm diffused light source Detectors: Low-noise CCD. Thermoelectrically cooled Acquisition Times: Fluorescence (700 and 800 nm) channels: 30 s, 2 min, 10 min plus variable time feature Chemiluminescence channel: 30 s, 2 min, 10 min, 60 min plus variable time feature Focusing: Automatic Operating Conditions: For indoor use only; operating temperature C and dew point < 22 C, non-condensing; maximum operating temperature may be reduced at elevations above 2000 m Power Requirements: Universal input range is between VAC and VAC (voltage fluctuations not to exceed 10% of the nominal voltage); 50/60 Hz; at 120V, 4 Amp maximum; at 120V, 0.3 Amp typical when idle. Overvoltage Category II Dimensions (instrument only): 67.3 cm H 41.4 cm W 47 cm D ( " 18.5 ) Depth with imaging drawer open is 59.7 cm (23.5 ) Weight: 27 kg (60 lb) Odyssey was the only one that came close to accurately measuring 10-fold serial dilutions of a protein sample over 3 orders of magnitude. Robin Rylaarsdam, Benedictine University, Via SelectScience

18 Optimized Detection Chemistry Variables Reduced, Options Multiplied As you incorporate rigorous study designs into measurable experiments, you ll need assay chemistry solutions that are adaptable to your workflow. Enzymatic detection chemistries are difficult to control and optimize. Additionally, the inability to multiplex limits throughput and efficiency. Get excellent detection sensitivity and options to multiplex and normalize with near-infrared (NIR) fluorescence. LI-COR reagent products for quantitative Western blotting reduce variability in detection chemistry and improve throughput, facilitating adherence to data reporting guidelines. Normalization of signal intensity to total protein loading (assessed by staining membranes using Coomassie blue, Ponceau S or other protein stains) is preferred. House-keeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression. Instructions for Authors, Journal of Biological Chemistry Positive and negative controls, as well as molecular size markers, should be included on each gel and blot... Image Integrity: Authors and Referees, Nature Publishing Group - Nature; Author Guidelines, EMBO Molecular Medicine IRDye Fluorescence Substrate HRP Fluorescence direct detection: Stable Signal ECL indirect detection: Dynamic Signal Get consistent data with NIR fluorescence. Direct detection using stable NIR fluorescence signals yields consistent data without the variables of enzymatic reactions, like time of exposure, substrate type and availability. 18

19 Western Blotting Reagents Track and Measure Targets with Stable Chemistry Get sensitive detection and multiplexing options with IRDye Infrared Dyes and dye-conjugated products. IRDye fluorophore-conjugated secondary antibodies Odyssey and Chameleon molecular weight markers IRDye infrared fluorescent dyes Quantitate and Validate Your Way Measure protein expression changes with accuracy; whether you normalize targets to total protein loading or validated housekeeping proteins. REVERT Total Protein Stain Primary antibodies against housekeeping proteins Odyssey Loading Indicators for validating housekeeping proteins Enhance Signal-to-Noise Ratios for Efficient Quantification Minimize background autofluorescence and improve detection sensitivity with reagents optimized for near-infrared fluorescence imaging. Immobilon -FL PVDF and Odyssey Nitrocellulose Membranes Odyssey Blocking Buffer Protein Sample Loading Buffer 19

20 Reliable Data Analysis Original Results Untouched As you process experimental observations into conclusive outcomes, the data analysis software that you count on must maintain the integrity of your work. Software features not designed for Western blotting data analysis can introduce variables and/or unevenly affect results, eventually compromising accuracy. Get software designed for streamlined Western blot data analysis. LI-COR Image Studio software integrates data acquisition, analysis, and archiving within a simple interface, so you can eliminate variables in digital transfers, while securing your original data according to publishers recommendations. Our screening process examines whether adjustments of brightness, contrast, or color balance have been applied to the entire image and that adjustments do not enhance, erase, or misrepresent any information present in the original, including the background. Editorial Policies, Journal of Cell Biology All images submitted to The Journal of Immunology must accurately represent the original data. Original data (digital files, autoradiographs, films, etc.) for all experiments should be fully annotated, secured, and retrievable. Information for Authors, The Journal of Immunology

21 Image Studio Data Analysis Software One software program that simplifies data capture, analysis, and record keeping. LI-COR Image Studio software secures the integrity of your results, by keeping variables like binning and non-uniform adjustments out of the equation. Consistent image acquisition settings to minimize variability in data capture Analysis options to adjust, normalize, quantitate, and chart data, without affecting original data Convenient archiving of all raw data for documentation and future reference Acquire and Analyze Western blots Protein and DNA gels Plate-based assays Arrays Tissue sections Download a free version of Image Studio Lite at licor.com/analyze

22 Training Solutions Round Out Your Western Blot Training Whether you are just getting started with near-infrared Western blotting or looking for practical guidance, you have an expert to coach you and your lab members through every step of the process; from protocol optimization to data reporting. Through our on-site new-user and personalized advanced training programs, you will learn: Tips on experimental design, and optimization and validation of reagents Best practices for imaging and data capture Data analysis methods including quantification, normalization, and presentation Techniques to optimize cell-based protein assays and other applications Methods to meet current Western blot publishing guidelines An online training center for self-paced learning, Lambda U presents a variety of educational resources, including: Course series on experimental design and workflows Standardized protocols and optimization guidelines Video tutorials and technical resources for Western blotting best practices Sign up for a course at lambdau.net 22

23 Support Resources Your Questions Answered Having a bad blot day in the lab? Not sure how and where to start troubleshooting? Need a pep talk before your next experiment? You will always have us by your side, just a call or a click away. Take advantage of direct access to: Our scientists, engineers, and support team. Worldwide. Through , phone, chat, or social. A wealth of educational resources, including protocols, handbooks, tutorial videos, webinars, and best practices. For all your immunoassay needs. Find Western Blot Answers at licor.com/qwbresources 23

24 Biological Assay Services End-to-End Protein Assay Services for Your Project Needs There are many demands on your time, resources, and expertise. Could you use some collaborative guidance? Entrust your protein assay experiments to the LI-COR Custom Services team. For your protein assay projects, ask us about: Experimental design, protocol development and imaging Reagent validation and optimization Data quantification, normalization, and analysis Confirmatory studies Higher-throughput analysis For a free consultation, go to licor.com/bioassays 24

25 Near-Infrared Fluorescence Western Blot Chemiluminescent Western Blot Tissue Section Imaging Coomassie Protein Gel Documentation In-Cell Western Assay Protein Arrays 25

26 References 1. Prinz F, Schlange T, Asadullah K Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov 10, doi: /nrd3439-c1 2. Begley GC, Ellis LM Drug development: Raise standards for preclinical cancer research. Nature 483, doi: /483531a 3. Vasilevsky NA, Brush MH, Paddock H, et al. On the reproducibility of science: unique identification of research resources in the biomedical literature. Abdullah J, ed. PeerJ. 2013;1:e148. doi: /peerj Freedman LP, Cockburn IM, Simcoe TS (2015). The Economics of Reproducibility in Preclinical Research. PLOS Biology 13(6): e journal.pbio Baker M. 1,500 scientists lift the lid on reproducibility. Nature (News Feature) 533, How Can Scientists Enhance Rigor in Conducting Basic Research and Reporting Research Results? American Society for Cell Biology. Web. wp-content/uploads/2015/11/how-canscientist-enhance-rigor.pdf. Accessed October 6, A Framework for Ongoing and Future National Science Foundation Activities to Improve Reproducibility, Replicability, and Robustness in Funded Research. December National Science Foundation. Web. gov/attachments/134722/public/ Reproducibility_NSFPlanforOMB_ Dec31_2014.pdf. Accessed October 6, Enhancing Reproducibility through Rigor and Transparency. National Institutes of Health. Web. guide/notice-files/not-od html. Accessed October 6, Reproducibility and Reliability of Biomedical Research. The Academy of Medical Sciences (UK). Web. acmedsci.ac.uk/policy/policy-projects/ reproducibility-and-reliability-ofbiomedical-research. Accessed October 6, Enhancing Research Reproducibility: Recommendations from the Federation of American Societies for Experimental Biology. Federation of American Societies for Experimental Biology. Web. and-communications/science-policy-and- Research-Issues/Research-Reproducibility. aspx. Accessed October 6,

27 11. Improving the reproducibility of biomedical research: a call for action. The Interacademy Partnership for Health. Web. Accessed October 6, A call for action to improve the reproducibility of biomedical research. The Interacademy Partnership for Health. STATEMENT%20ON%20RESEARCH%20 REPRODUCIBILITY.pdf. Accessed October 6, Freedman LP, Venugopalan G and Wisman R. Reproducibility2020: Progress and priorities [version 1; referees: 2 approved]. F1000Research 2017, 6:604 (doi: / f1000research ) 16. Reproducibility2020. The Global Biological Standards Institute. Web. gbsi.org/work/reproducibility2020/. Accessed October 6, Transparency and Openness Promotion (TOP). Center for Open Science. Web. Accessed October 6, Recommendations to Funding Agencies for Supporting Reproducible Research. American Statistical Association. Web. POL-ReproducibleResearchRecommendati ons.pdf. Accessed October 6, DFG Statement on the Replicability of Research Results. The Deutsche Forschungsgemeinschaft (DFG - German Research Foundation). Web. research-in-germany.org/en/researchlandscape/news/2017/04/ dfgstatement-on-the-replicability-of-researchresults.html. Accessed October 6,

28 Take the next step toward meeting the changing needs of research reporting. Start a conversation. licor.com/consult LI-COR Biosciences 4647 Superior Street Lincoln, NE Phone: Toll free: biosales@licor.com LI-COR Distributor Network: Regional Offices LI-COR Biosciences GmbH Siemensstraße 25A Bad Homburg Germany Phone: +49 (0) bio-eu@licor.com LI-COR Biosciences UK Ltd. St. John s Innovation Centre Cowley Road Cambridge CB4 0WS United Kingdom Phone: +44 (0) bio-eu@licor.com LI-COR, Odyssey, Data Integrity, IRDye, Image Studio, In-Cell Western, FieldBrite, Chameleon, REVERT, and Lambda U are trademarks or registered trademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respective owners. For patent information, visit LI-COR, Inc. 11/ The LI-COR board of directors would like to take this opportunity to return thanks to God for His merciful providence in allowing LI-COR to develop and commercialize products, through the collective effort of dedicated employees, that enable the examination of the wonders of His works. Trust in the LORD with all your heart and do not lean on your own understanding. In all your ways acknowledge Him, and He will make your paths straight. Proverbs 3:5,6