Collecting and presenting data
Collecting and presenting data
These guidelines do not presuppose that there is such a thing as a perfect experiment or an ironclad conclusion. Indeed, variation between studies and experiments is an important source of new discoveries. For this reason it is important to report data and experimental conditions as fully and transparently as possible both to verify findings and also to help future researchers identify sources of variation and anomalies for generating new hypotheses.
Depending on the research question being asked or experimental system being used, some of the following “best practices” may not be feasible or appropriate for your study. In that case, exercise the “best practice” of explaining and justifying any deviation from the norm.
Your gels and blots should show appropriate context: Gels and blots should not be excessively cropped, any spliced-out lanes should be clearly marked, and positions of any molecular weight markers should be clearly indicated.
N values, including those for technical and biological replicates, should be clearly stated and explained. Whenever possible, use scatter plots rather than bar graphs.
Clearly indicate what any error bars represent (standard deviation, standard error of the mean, or confidence interval).
Make sure that any quantification method used, including densitometric quantification of images, is within the linear range afforded by that method.
Images should be of sufficient quality and resolution.
Descriptions of biological materials
Papers should describe biological materials in enough information to uniquely identify materials, including repository accession numbers when available. Consider using Research Resource Identifiers (RRIDs), unique searchable identifiers, to report which reagents and tools were used. Make sure to keep track of the following information as you perform experiments.
Antibodies. Include sources, dilutions, and validation criteria.
Define species of origin and source of all antibodies used, including catalog/lot numbers.
Describe how novel antibodies were generated, including preparation and purification of epitope/antigen.
Describe data supporting antibody specificity, including post-translational modifications or neoepitopes.
If possible, demonstrate loss of immunoreactivity following genetic or other molecular modification to the antigen.
Cell lines. Include information about cell line source, derivation, authentication, and contamination (such as mycoplasma) status.
Cell lines should be checked against the ICLAC commonly misidentified or contaminated cell lines database.
Specify method used to authenticate cell lines.
Animals. Specify animal source, species, strain, sex, age, and relevant details of husbandry. For transgenic animals, specify the genetic background.
Quantitative data and statistics
Quantitative data must be reported transparently to ensure reproducibility and enable discovery. Find more information about the rationale for these recommendations in the Editorial: Transparency is the Key to Quality.
Clearly define replicates. How many technical and biological replicates were performed during how many independent experiments? How is the replication represented by the data points shown in figures? Report this information in the methods section and include relevant details in figure legends.
If you choose to show representative data from several independent experiments or assays, please indicate where the remaining data can be found.
If a well-established and supported community database where you can upload your data does not already exist, ensure the long-term availability of your data by depositing it in a database such as Zenodo, Dryad, or Figshare and report the corresponding doi.
Excluded data. State whether any data were excluded. If so, indicate the reason and criteria for exclusion.
Individual data points. Whenever possible (especially in results with fewer than 30 data points), show data as individual points to make the distribution and variation clear.
Randomization. Indicate whether and how samples were randomized during analysis and processing.
Reporting experimental variability. The standard deviation (SD) or confidence intervals (CI) should be used to report the variation/precision of each data set, with appropriate documentation.
Testing for statistically significant differences. Tests used to determine whether differences between data sets are statistically significant should be precisely and fully described. Comparisons of more than 2 data sets from the same experiment should be done with an appropriate 1- or 2-way ANOVA, with a complete reporting of all results from these tests.
Image preparation begins when you collect your data, and it’s worth your time to get this right from the outset. Questions regarding image validity can lead to delays in publication, corrections to published articles, requests to withdraw manuscripts, or retractions of articles.
Read Data Integrity Manager’s Kaoru Sakabe’s Due Diligence columns for detailed advice and commentary on figure preparation.
No specific feature within an image may be enhanced, obscured, moved, removed, or introduced. Blemishes, stray marks, and so forth are a hallmark of true data.
Adjustments of brightness, contrast, or color balance are acceptable if they are applied to every pixel in the image and as long as they do not obscure, eliminate, or misrepresent any information present in the original, including the background. Nonlinear adjustments (e.g., changes to gamma settings) must be disclosed in the figure legend.
Do not prepare images in PowerPoint or save them in JPEG format.
If scanning images with a flat-bed scanner, scan with a minimum resolution of 300 dpi with no adjustments.
Microscopy data should be collected and presented with a magnification necessary to clearly illustrate the findings and include scale bars.
Record the following information regarding microscope image acquisition:
Make and model of microscope
Type, magnification, and numerical aperture of the objective
Camera make and model
Any software used for image processing subsequent to data acquisition. Please include details and types of operations involved (e.g., type of deconvolution, 3D reconstitutions, surface or volume rendering, gamma adjustments, etc.)
If you export files from a microscope or other acquisition device, be sure to use consistent file formats (8 bit, 16 bit, etc.).
Micrographs should include a larger field of view at a lower magnification and an inset at a higher magnification that shows features/regions of interest.
Images that are a composite of separate images should have borders of original images clearly marked.
Co-localization of two or more signals from different fluorophores or stains should be supported by merged images from the channels at a resolution sufficient to distinguish the features of interest.
Fluorescence images should show signals from individual channels in gray scale to reveal the full dynamic range of intensities, and to allow color-blind individuals to appreciate your data. Merged images should be presented in color, with distinct colors for individual channels.
Quantitative statements regarding the cellular distribution of molecules or changes in their levels should be supported by quantification of corresponding regions.
When quantifying, make sure the exposure used results in a signal that is within the linear range afforded by that method.
In the case of fluorescence images using primary antibody and secondary antibody combinations, controls such as a non-immune antibody, omission of the primary antibody, and absence of antigen may be necessary to demonstrate specificity.
Criteria for image selection and analysis should be clearly explained, and data should include numbers of replicates and appropriate statistical analyses to determine significance.
Blot images and quantification
Gels provide a perfect blank canvas to test a variety of scientific questions, but the meaning behind those bands is lost without proper context. Follow the guidelines below to make sure your results are clear and convincing. See this presentation by Associate Editor Roger Colbran for more information.
Blots should show full tonal range. A loss of tonal range is a loss of data.
Crop immunoblots in a way that retains information about antigen size and antibody specificity.
Include positions of molecular weight markers above and below the band(s) of interest. (Example below.)
Avoid assembling figures of blots by splicing lanes from different sections of a gel. If blots must be spliced, borders must be clearly marked and explained in the figure legend (see example below).
Record 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.
Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible. “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression.
Phospho-specific antibody signals should be normalized to total levels of the target protein.
Structures and models
Data for new NMR spectroscopy, X-ray crystallographic or cryo-electron microscopy structures should always be deposited in the Protein Data Bank (NMR and X-ray) or cryo-EM depositories (e.g. https://www.ebi.ac.uk/pdbe/emdb/).
All chemical shifts for backbone and side chain assignments (as well as methyl group chemical shift assignments) must be deposited in the BMRB.
For JBC papers, structural coordinates describing experimentally determined new structure determinations must be submitted to the Protein Data Bank and BMRB and released immediately at acceptance, because publication will occur within 24 hours of manuscript acceptance. No data is to be withdrawn from the Protein Data Bank or the BMRB once a paper has been accepted and published as a Paper in Press article.
Papers describing the activity of synthetic or natural chemical entities must include the chemical structures of such molecules, either as systematic names or as drawn structures.
For synthetic chemicals, a synthetic protocol should be provided, generally as part of the supporting information. Alternatively, reference to a publication or issued patent that includes such a synthetic scheme and details can be provided.
For natural chemicals, methods for extraction/purification of the chemical and determination of its structure should be provided in the methods section.
Enzyme activity data
Papers reporting kinetic and thermodynamic data should include the identity of the biomolecule(s), relevant biological information (e.g., species and tissue normally found in, any post-translational modifications), preparation and criteria of purity, assay conditions, methodology, activity, and all other information relevant to judging the reproducibility of the results. The guidelines from the Beilstein Institut/STRENDA Commission have more details and suggestions to keep in mind as you are performing experiments and preparing your manuscript.
Enzyme activity (steady-state) generally should be reported in terms of kcat (Vmax divided by molar enzyme concentration); Vmax per time is also acceptable. Km units are given in molarity.
Any other units of activity (absorbance, % change) should be converted to units of molarity to express kcat or Vmax. Values of kcat (Vmax) and Km should be estimated using nonlinear fitting (and the software system cited).
Parameters should include estimates of error (e.g., SD). The use of linear transformations for calculation of Michaelis-Menten parameters is recognized to be inaccurate. The use of any linear transformations should be justified (e.g., graphical presentation of inhibition).
A lack of activity should be defined in terms of a limit of detection. In a series of comparisons to a basal or control level of activity (e.g., set as unity or 100%), this activity should be indicated, in the units mentioned above, along with estimates of error. The inclusion of examples of some of the raw data is encouraged.
Ki values are preferred to IC50.
Datasets can be submitted to the STRENDA database, which is an electronic validation and storage system for functional enzyme data and provides a valuable search repository for standardized enzyme kinetics data, which can benefit a larger research community. If an author chooses to submit kinetic data to STRENDA DB, the data become publicly available only after the corresponding article has been published. Additionally, authors can report new functional results to Uniprot, a resource of protein sequence and functional information, so the description of their protein of interest is up to date.
Authors of papers that include proteomics data should comply with the guidelines developed by Molecular and Cellular Proteomics.
Report animal source, strain, sex, age, and details of husbandry.
State whether studies were blinded or not. If so, indicate the method and extent of blinding.
Ensure that all research has been reviewed and approved by an Institutional Animal Care and Use Committee and include a statement to this effect in the experimental procedures section of the manuscript. Consult the ARRIVE guidelines for reporting animal research.
All studies involving human subjects must be approved by the appropriate review board(s) and abide by the Declaration of Helsinki principles; include a specific statement declaring approval and Helsinki compliance in the experimental procedures section. Do not provide any identifying information (e.g., names, true initials, recognizable images) unless the information is essential for scientific purposes and the patient (or patient’s parent/guardian) gives written informed consent for publication. If the patient is deceased, then the authors should seek consent from a relative. For submission to JBC, upload a completed consent form as additional Supporting Information for review only.
Gene expression and genetic manipulation
Include appropriate controls for CRISPR/Cas experiments, such as results from two or more guides and two or more independent clones. Experiments in cells, including induced pluripotent stem cells, should include an isogenic control with cells that retain a wild-type allele.
Verify CRISPR/Cas-mediated insertion/deletions by sequencing and show loss of the protein by immunoblotting, if antibodies are available. When antibodies are not available, changes in RNA expression should be validated by quantitative RT-PCR.
RNA interference (RNAi) experiments should include at least two different siRNAs that are complementary to different portions of the target gene and control siRNAs with sequences altered from the target.
Significant depletion of target gene expression by siRNAs should be demonstrated by measurements of target protein or mRNA, and can also be addressed by rescue experiments involving expression of target gene sequences refractory to siRNA.
Studies that assess the biological effects of changes in gene expression that are based on measures of RNA levels (e.g., RNA-seq, microarrays, QT-PCR) should also have supporting evidence to confirm that the changes in RNA levels have functional consequences.
Microarray data should be deposited in a public database compliant with the MIAME guidelines (Minimum Information about a Microarray Experiment; see below), for example GEO or ArrayExpress. Information in bold should also be described in the methods section of the manuscript.
The raw data for each hybridization (e.g., CEL or GPR files)
The final processed (normalized) data for the set of hybridizations in the study (e.g., the gene expression data matrix used to draw the conclusions from the study)
The essential sample annotation including experimental factors and their values (e.g., compound and dose in a dose response experiment)
The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridizations are technical, which are biological replicates)
Sufficient annotation of the array (e.g., gene identifiers, genomic coordinates, probe oligonucleotide sequences or reference commercial array catalog number)
The essential laboratory and data processing protocols (e.g., what normalization method has been used to obtain the final processed data)
Appropriate FACS analysis procedures should include multiple parameters and/or time points to establish biological states.
See also recommendations from the International Society for Advancement of Cytometry regarding Minimum Information About a Flow Cytometry Experiment.