Writing the Results Section Data Analysis and Interpretation BIOL502

Assessment 2

Writing the Results Section

In the results section, you will present the following results:

1. Flow cytometry plots. You have been provided with a powerpoint file (PARRA Flow Cytometry Figures) showing two unstimulated and two stimulated samples. Each sample is presented as 4 plots. Some information about the plots is provided on the right-hand side of each slide. The plots can be directly copy-pasted into your report. You may include all 4 samples, or just one each of the stimulated and unstimulated samples. You may present them as a single figure or two or more figures. Each figure must have an accompanying figure legend that describes what is being shown. A text that accompanies the figures will interpret the figures. You are expected to include in the text accompanying the figures some explanation of the importance of FSC Vs SSC, as well as FSC-H Vs FSC-A (doublets Vs singlets) and compare and contrast the % of cells identified as CD69+ or IFNg+ in each sample. For a nice description for discriminating between singlets and doublets, see: https://www.bio-rad-antibodies.com/flow-cytometry-doublet-discrimination.html.
2. Quantification of flow cytometry data. This is an excel file that includes three tabs: i) Flow cytometry data showing the % of CD69+ or IFNg+ samples collected from classes at Parramatta; ii) Plotting the data. In this sheet is provided example data, as well as an image of how the data should be visualised. The difference between the two samples must be analysed using the non-parametric Mann-Whitney rank-sum test. Non-parametric tests are used in place of parametric tests (e.g. ANOVA) is the data set is small, does not follow a normal distribution, and the paired samples have unequal variance. iii) The Mann-Whitney explained tab provides a link to an online tool that will calculate the Mann-Whitney p-value for you. There is additional information showing how the Mann-Whitney test is calculated (it is not difficult).
3. qPCR analysis of RNA expression. As explained in the practical class, you will use the 2^-ddCt method to calculate the fold change in RNA between unstimulated and stimulated cells. You will analyse the results using the two-factor ANOVA with replication. The results of this test will be used to interpret the results of your analysis in terms of whether there was an increase in expression between stimulated and unstimulated cells, and whether this changed with time.
4. DNA methylation MSRE assay. As explained in the proactical classes, you are expected to calculate the dCt value for HpaII-undigested and MspI-undigested for each sample. You need to remember the difference in the activity of MspI and HpaII towards methylated an unmethylated DNA, and consider what the difference is in PCR amplification if the genomic DNA template is left undigested or digested with MspI (independent of DNA methylation) or HpaII (methylated Vs unmethylated DNA), and how this relates to differences in Ct value in a qPCR reaction.

Important points to remember.

• In the brief introduction you will set the scene, describing the rationale for why the experiments were done. You carried out a functional investigation of gene expression at a locus that includes disease-associated SNPs identified in GWAS. Why is IFN-g important?
• Similar to Assessment 1, you will be expected to integrate the information presented in each of the figures to reach a conclusion
• To do this, you should understand the hypothesis: what is it you want to test (discover)? How do each of the different assays contribute to testing the hypothesis?
• It is a good idea to start your results section by stating your hypothesis: In this report we set out to test. To do this we first investigated
• Think carefully about the order of your figures it should create a narrative (story) that explains how you are testing your hypothesis
• Working in groups to discuss what the different experiments mean and how they fir together to test a hypothesis is a god idea: just make sure you then split up and write your reports
• Do NOT copy-paste the ANOVA results directly into your report: look at the outcomes and p-values and incorporate these results into the test that interprets the figures
• Your conclusion should be related directly to what you found. It is important to first consider your hypothesis what was the outcome of your experiments? I have provided 3 research papers that should assist you in writing your conclusion