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Data Presentation: the importance of transparency
Roger J. Colbran
Associate Editor
Credit: Amanda Fosang, Associate Editor
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Why Improve Transparency?
• Irreproducibility of pre-clinical studies
• Negative impact on political and public will to fund research…...
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Reason for irreproducibility
• Poor methodological descriptions
• Reagents not described adequately and/or validated – antibodies, siRNAs, small molecules
• Inappropriate data interpretation
http://www.nih.gov/research-training/rigor-reproducibility
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Efforts Toward a Solution…...Improve transparency of JBC papers by:
1. Improving description of methods, including reagents
2. Clearly defining reproducibility (technical and biological replicates)
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1. Biological MaterialsInstructions to Authors: Descriptions of biological materials should include enough information to uniquely identify materials, such as: • Repository accession numbers when available• Antibodies - source, dilutions, and validation criteria• Cell lines - source, authentication, derivation, and contamination (such as mycoplasma) status
• Animals - source, species, strain, sex, age, husbandry• Transgenic animals – genetic background
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2. Experimental uncertainty, statistics
"There are three kinds of lies: lies, damned lies, and statistics."
Attr. (by Mark Twain)Benjamin Disraeli, British Prime Minister
Transparency is the key…....
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2. Experimental uncertainty, statisticsJBC Instructions to Authors:• Authors must include information on uncertainty and reproducibility of data in figure legends.
• Authors must state numbers of independent samples (biological replicates) and replicate samples (technical replicates) analyzed and report how many times each experiment was repeated.
• Variation/precision should be reported by standard deviation (SD) (preferable), confidence intervals (CI) or standard error of the mean (SEM).
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2. Experimental uncertainty, statistics
Motulsky, 2014. J. Pharmacol. Exp. Ther. 351:200
Same data analyzed and plotted differently:
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2. Experimental uncertainty, statistics
Instructions to Authors: Additional issues for animal studies
• State whether or not animals and/or samples analyzed were randomized, and indicate how.
• State whether studies were blinded or not. If so, indicate the method of blinding.
• State whether any data were excluded. If so, indicate the reason and/or criteria for exclusion.
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3. Graphical presentation of data
Instructions to authors:• Scatter plots strongly recommended for small data
sets (can overlay a mean ± SEM on top of the individual data points);; box and whisker plots for large data sets.
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3. Graphical presentation of data
Weissgerber et al., 2015. PLOS Biology 13: e1002128
Mean ± SEM bar graphs can be misleading
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4. Western blotsInstructions to authors:• Define species of origin and source of all antibodies used, including catalog/lot numbers.
• Describe how novel antibodies were generated, including preparation/purification of epitope/antigen.
• Describe data supporting antibody specificity, including post-translational modifications or neoepitopes.
• If possible, demonstrate loss of immunoreactivity after genetic or other molecular modification to the antigen.
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4. Western blots
Instructions to authors:• Immunoblots should be cropped in a way that retains information about antigen size, antibody specificity.
• Cropped images should include positions of MW markers above AND below the band(s) of interest.
• Avoid assembling figures of blots by splicing lanes from different sections of a gel. If they must be spliced, borders must be clearly marked and explained in the figure legend.
Keep original data and unprocessed scans!
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4. Western blotsInstructions (semi-quantitative analyses):• Explain how data were obtained, whether signal intensity was linear with antigen loading, and how protein loading was normalized. Some detection methods (e.g., ECL) have a very limited linear range.
• Strongly prefer normalizing signal intensity to total protein loading (assessed by staining membranes for total protein). “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression.
• Phospho-(or other PTM-) specific antibody signals should be normalized to total levels of target protein.
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4. Western blots
Gustin et al., 2010. Neurobiol Disease 39:283–291
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4. Western blots
Instructions to authors:During editorial review, authors may be asked to provide high resolution images of original immunoblots, quantification details, and antibody validation data.
Keep original data and unprocessed scans!
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Implementation1. Clearly define expectations and policies
2. Author education
3. Consistency in reviewsEditorial policies: http://www.jbc.org/site/misc/edpolicy.xhtml
Instructions to authors: http://www.jbc.org/site/misc/ifora.xhtml