Rigor and Reproducibility

  Eight steps to Rigorous and Reproducible Experiments in Biomolecular Research at UNC:

1. If using a core facility, consult with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.
2. Design your experiment with sufficient controls (rigor) and replicates (reproducibility)
3. Assure that ALL of your reagents (antibodies, cell lines, mice) are fully validated (see below)
4. Have a clear and detailed protocol (SOP) and data analysis plan. Assure that the protocol is strictly followed or that any deviation is well documented.
5. Assure that the staff or students performing the experiment are well trained and understand each step and the importance of performing them precisely.
6. Use only well-maintained instrumentation, preferably maintained and operated in a core facility with expert staff (see #1 above).
7. Document all steps, reagents, equipment and data analysis methods used in the experiment. Assure that the both the documentation and the data itself are properly stored in a safe data management repository.
8. Acknowledge the Cancer Center Support Grant (P30 CA016086) (if applicable), other grants that support the core, the core (by name), and core staff in publications.

Guide to Rigor and Reproducibility for the UNC Mass Cytometry Core

1. Robust mass cytometry results begin with a strong experimental design. Work with our experts early in the design process to discuss your goals. We can help you build strong experiments by suggesting options such as internal spiked controls and barcoding to control and monitor batch effects.
2. Please see ‘Resources’ for best practice protocols and sample handling.
3. Unless using a pretitered commercial kit, all antibodies and reagents (such as Iridium and viability stains) should first be titered for optimal staining concentration and validation. The Mass Cytometry Core staff will help with this process.
4. Design your experiment with sufficient controls (rigor) and replicates (reproducibility). Consult a statistician for help developing a Power Analysis.
5. Sites to help with proper reagent validation:

• • NIH FAQs on Rigor and Reproducibility
  • Antibody Identification Database
  • Special Edition of Cytometry Part A, focused on Rigor and Reproducibility
  • Uhlen et al., A Proposal for Validation of Antibodies, Nature Methods (2016)

6. To ensure and document optimal instrument performance on a daily basis and prior to data acquisition the UNC mass cytometer:

• • is tuned to manufacturer’s specifications
  • has background signals recorded
  • is performance verified using control (EQ) beads.

7. Bead normalized fcs files will be uploaded to dated folders for each individual researcher on UNC’s Cytobank Enterprise platform. In addition, all data files (FCS, fcs and imd) will be stored on the instrument’s hard drive for 90-days. After 90-days, the FCS, fcs and imd files will be zipped and stored remotely on LCCC servers for 5 years. After 5 years, all files are deleted. If possible, researchers will be contacted at this time and will be given the option to personally store their data files.
8. If you use UNC Mass Cytometry Core-generated data in your publications, please remember to cite the following:

• • University of North Carolina Mass Cytometry Core (Marie A. Iannone, Facility Director)
  • University Cancer Research Fund (UCRF)
  • UNC Cancer Center Core Support Grant #P30CA016086