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Preprint

Statistical analysis

DC David Chen
JW Josephine Wu
GV Gordana Vunjak-Novakovic

Last updated date: May 9, 2021 Views: 863 Forks: 0

original An abbreviated version of this protocol was published in Science Translational Medicine in Oct, 2020
Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration
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  1. Open GraphPad Prism software. This protocol was written based on GraphPad Prism 8.
  2. Create a data table.
    NEW TABLE & GRAPH > Grouped > Data table: Enter or import data into a new table > Options: Enter and plot a single Y value for each point > Create
  3. Enter group names (e.g. Native, Cartilage-bone, Bone-only, Acellular) and data for a single assay. This data table will be referred to as Data X in the subsequent steps. For each assay, create a separate data table.
    Data Tables: + New Data Table…
  4. Native data, which were expected to be normally distributed, were checked for outliers using the robust regression and outlier removal method and a Q coefficient of 1% to limit the false discovery rate.
    Data Tables: Data X > Analysis: Analyze > Column analyses: Identify outliers > Analyze which data sets? Native > OK > Method: ROUT (recommended; can find any number of outliers) > How aggressive? Q = 1% > OK
  5. View outliers within the native data.
    Results: Identify outliers of Data X > Outliers
  6. Remove outliers within the native data.
    Data Tables: Data X > remove values identified in previous step.
  7. If n>4 in each group, check for whether they fit Gaussian distributions using the Shapiro-Wilk normality test.
    Data Tables: Data X > Analysis: Analyze > Column analyses: Normality and Lognormality Tests > Analyze which data sets? Select all > OK > Which distribution(s?) to test? Normal (Gaussian distribution) > Methods to test distributions(s): Shapiro-Wilk normality test > Calculations: Significance level (alpha): 0.05 > OK
  8. View results of normality test.
    Results: Normality and Lognormality Tests of Data X > Passed normality test (alpha=0.05)?
  9. If n≥4 and normality test was passed, carry out statistical analysis by one-way analysis of variance (ANOVA) test and post hoc Tukey-Kramer’s comparison test to compare means between groups.
    Data Tables: Data X > Analysis: Analyze > Column analyses: One-way ANOVA (and nonparametric or mixed) > Analyze which data sets? Select all > OK > Experimental design: No matching or pairing > Assume Gaussian distribution? Yes. Use ANOVA. > Assume equal SDs? Yes. Use ordinary ANOVA test. > Multiple Comparisons > Followup tests: Compare the mean of each column with the mean of every other column. > Options > Multiple comparisons test: Correct for multiple comparisons using statistical hypothesis testing. Recommended. > Test: Tukey (recommended). > Multiple comparisons options: Report multiplicity adjusted P value for each comparison. Family-wise alpha threshold and confidence level: 0.05 (95% confidence interval) > OK
  10. If n<4 or normality test was not passed, carry out statistical analysis by nonparametric Kruskal-Wallis test and post hoc Dunn’s multiple comparison test to compare the means between groups.
    Data Tables: Data X > Analysis: Analyze > Column analyses: One-way ANOVA (and nonparametric or mixed) > Analyze which data sets? Select all > OK > Experimental design: No matching or pairing > Assume Gaussian distribution? No. Use Nonparametric test. > Multiple Comparisons > Followup tests: Compare the mean rank of each column with the mean rank of every other column. > Options > Multiple comparisons test: Correct for multiple comparisons using statistical hypothesis testing. Recommended. Test: Dunn’s. > Multiple comparisons options: Report multiplicity adjusted P value for each comparison. Family-wise alpha threshold and confidence level: 0.05 (95% confidence interval) > OK
  11. View results of ANOVA or Kruskal-Wallis tests.
    Results > Ordinary one-way ANOVA of Data X or Kruskal-Wallis test of Data X > Multiple comparisons > Summary
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Chen, D, Wu, J and Vunjak-Novakovic, G(2021). Statistical analysis. Bio-protocol Preprint. bio-protocol.org/prep1068.
  2. Chen, D., Wu, J. Y., Kennedy, K. M., Yeager, K., Bernhard, J. C., Ng, J. J., Zimmerman, B. K., Robinson, S., Durney, K. M., Shaeffer, C., Vila, O. F., Takawira, C., Gimble, J. M., Guo, X. E., Ateshian, G. A., Lopez, M. J., Eisig, S. B. and Vunjak-Novakovic, G.(2020). Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration . Science Translational Medicine 12(565). DOI: 10.1126/scitranslmed.abb6683

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