20.109(S09):Module 2

20.109(S09): Laboratory Fundamentals of Biological Engineering

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Protein Engineering Expression Engineering Cell-Biomaterial Engineering

Module 2

Instructors: Leona Samson, | Agi Stachowiak

TA: | Steve Goldfless

Additional development and instruction: Peter Svensson (Samson lab, mouse gene siRNAs) and Stuart Levine (BioMicro Center, microarray analysis). Original module developed by [1] and Leona Samson.

In this experiment, we will consider direct and indirect effects of an experimental perturbation, specifically the use of RNA interference (RNAi). Each group will test two different short interfering RNAs (siRNAs) in mouse cells. We will first compare the efficacies of six different siRNAs targeting luciferase. Knockdown of this gene is readily measured using a luminescence assay and reporter plasmid. Next we will test four different commercially available siRNAs targeting mouse genes of interest. Each siRNA should knock down the gene of interest (direct effect), but is also likely to affect other genes in pathways involving the target gene (indirect effect), and may even affect expression of genes that are unrelated to the target due to some non-specific effects of the siRNA or unintentional targeting of similar sequences (off-target effect). You will perform a genome-wide expression microarray experiment and try to assess how the changes in gene expression that you see fall into these three categories. Along the way, you will learn such valuable skills as how to culture cells, do basic statistics, and analyze large data sets.

Control Microarray Sample expression microarray spanning entire human genome, using identical RNA labeled with two different colours. The resulting equal red and green fluorescence intensities combine to produce yellow spots.