Model the data

1. Model the data

Now that you've pre-processed the data, it's time to perform the hypothesis tests.

2. Ready for analysis

After the pre-processing steps, you learned that the samples cluster in 3 main groups. The wild type mice are separated by treatment, but the Top2b null mice form one large cluster. This supports the hypothesis that Top2b null mice are resistant to the cardiotoxic effects of doxorubucin. To formally test this, you will perform a differential expression analysis with limma.

3. Steps for differential expression analysis

To do this, you will follow the same steps as before. First you'll build the design matrix, then construct the contrasts matrix, and finally test the contrasts.

4. Group-means model for doxorubicin study

Using the group-means parametrization, you will create a linear model with one coefficient for each of the 4 groups. Each coefficient models the mean expression level of the gene in the 3 replicates of that group. For example, beta-one measures the mean expression level in Top2b null mice treated with doxorubicin.

5. Contrasts for doxorubicin study

To investigate the mechanism of doxorubucin-induced cardiotoxicity, you will test 3 contrasts. First, the response of wild type mice to doxorubcin treatment. Second, the response of Top2b null mice to doxorubicin treatment. And finally, the interaction between genotype and treatment, or the differences between Top2b null and wild type mice in their response to doxorubicin treatment. Recall that the interaction term is constructed by contrasting the response to treatment in each genotype separately.

6. Testing the doxorubicin study

Lastly, you'll perform the hypothesis testing using the limma pipeline. However, this time when summarizing the results, you will also create a Venn Diagram with the limma function `vennDiagram`. This will reveal how many of the differentially expressed genes are shared between the 3 contrasts.

7. Let's practice!

Now it's time for you to test if Top2b has an affect on the mechanism of doxorubicin in heart cells.

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